Fusogenic lipid nanoparticles for target cell-specific production of a therapeutic protein fusogenic lipid nanoparticles and methods of manufacture and use thereof for the target cell-specific production of a therapeutic protein and for the treatment of a disease

ABSTRACT

Provided nucleic acid-based expression construct for the target cell-specific production of a therapeutic protein, such as a pro-apoptotic protein, within a target cell, including a target cell that is associated with aging, disease, or other condition, in particular a target cell that is a senescent cell or a cancer cell. Also provided are formulations and systems, including fusogenic lipid nanoparticle (LNP) formulations and systems, for the delivery of nucleic acid-based expression constructs as well as methods for making and using such nucleic acid-based expression constructs, formulations, and systems for reducing, preventing, and/or eliminating the growth and/or survival of a cell, such as a senescent cell and/or a cancer cell, which is associated with aging, disease, or other condition as well as methods for the treatment of aging, disease, or other conditions by the in vivo administration of a formulation, such as a fusogenic LPN formulation, comprising an expression construct for the target cell-specific production of a therapeutic protein, such as a pro-apoptotic protein, in a target cell that is associated with aging, disease, or other condition, in particular a target cell that is a senescent cell or a cancer cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. non-provisional patent application is a continuation of U.S.patent application Ser. No. 16/388,775, filed Apr. 18, 2019, whichclaims the benefit of U.S. Provisional Patent Application 62/659,676,filed Apr. 18, 2018, and U.S. Provisional Patent Application 62/821,084,filed Mar. 20, 2019, each of which is incorporated herein by referencein its entirety.

SEQUENCE LISTING

The instant application includes a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Nov. 29, 2022, isnamed 54636_707_301_SL.xml and is 65,524 bytes in size.

BACKGROUND OF THE DISCLOSURE Technical Field

The present disclosure relates, generally, to the field of medicine,including the treatment of disease, promotion of longevity, anti-aging,and health extension. More specifically, this disclosure concernscompositions and methods for reducing the growth and/or survival ofcells that are associated with aging, disease, and other conditions.Provided are expression constructs for target cell specific expressionof therapeutic proteins, which constructs exploit unique intracellularfunctionality, including transcription regulatory functionality, that ispresent within a target cell but is either absent from or substantiallyreduced in a normal, non-target cell. Such expression constructs areused in systems that include a vector for the delivery of a nucleic acidto a target cell, which vectors may comprise, but do not necessarilyrequire, a fusogenic lipid nanoparticle and, optionally, a targetingmoiety for enhancing the delivery of an expression construct to a targetcell.

Description of the Related Art

Cancer cells, senescent cells, and other cells having an undesirablephenotype can accumulate over the course of a person's life and, withoutappropriate treatment, such cells can contribute to or even cause aperson's morbidity and, ultimately, mortality.

The role of senescent cells in disease and the potential benefits ofeliminating senescent cells has been discussed in scientificpublications such as Baker et al. Nature 479:232-6 (2011). Systems andmethods have been described that purport to address the problem ofaccumulating senescent cells. For example, Grigg, PCT Patent PublicationNo. WO 1992/009298, describes a system for preventing or reversing cellsenescence with chemical compounds similar to carnosine and Gruber, U.S.Patent Publication No. 2012/0183534, describes systems for killingsenescent cells with radiation, ultrasound, toxins, antibodies, andantibody-toxin conjugates, which systems include senescent cell-surfaceproteins for use in targeting of therapeutic molecules.

The selective killing of senescent cells has proven impractical inmammals other than genetically-modified laboratory research animals.Currently-available systems and methods exhibit substantial systemictoxicity, inadequate targeting of cells of interest, and a lack ofadequate safety features. These shortcomings in the art have hamperedthe development of safe and effective therapies for the treatment ofcertain cancers and for slowing the effects of aging.

SUMMARY OF THE DISCLOSURE

The present disclosure is based upon the discovery that a cell, such asa cell that is associated with aging, a disease, and/or anothercondition (collectively, “a target cell”), can be selectively killed, ina target cell-specific manner, without the need for the targeteddelivery of a therapeutic agent to the target cell. The expressionconstructs, systems, and methods described herein overcome safety andefficacy concerns that are associated with existing technologies thatemploy targeted delivery of therapeutic agents, which technologies haveyielded limited therapeutic benefit to patients in need thereof.

As described herein, the present disclosure provides expressioncassettes, systems, and methods for inducing, in a target cell-specificmanner, the expression of a nucleic acid that encodes a protein that,when produced in a cell, reduces or eliminates the growth and/orsurvival of a cell, such as a cell that is associated with aging,disease, and/or other condition.

The expression cassettes, systems, and methods described herein exploitthe unique transcription regulatory machinery that is intrinsic tocertain cells that are associated with age (such as senescent cells),disease (such as cancers, infectious diseases, and bacterial diseases),as well as other conditions, which transcription regulatory machinery isnot operative, or exhibits substantially reduced activity, in a normalcell (i.e., “a non-target cell”) that is not associated with aging,disease, or other condition.

The presently-disclosed expression cassettes, systems, and methodsachieve a high degree of target cell specificity as a consequence ofintracellular functionality that is provided by, and unique to, thetarget cell, which intracellular functionality is not provided by, or issubstantially reduced in, a normal, non-target cell. Thus, the presentlydisclosed systems and methods employ nucleic acid delivery vectors thatare non-specific with respect to the cell type to which the nucleic acidis delivered and, indeed, the vectors described herein need not beconfigured for target cell-specific delivery of a nucleic acid (e.g., anexpression cassette) to achieve target cell specificity and,consequently, the therapeutically effective reduction, prevention,and/or elimination in the growth and/or survival of a target cell.

Within certain embodiments, the present disclosure provides expressionconstructs for the targeted production of therapeutic proteins within atarget cell, such as a cell that is associated with aging, disease,and/or another condition. The expression constructs disclosed hereincomprise: (1) a transcriptional promoter that is activated in responseto one or more factors each of which is produced within a target celland (2) a nucleic acid that is operably linked to and under regulatorycontrol of the transcriptional promoter, wherein the nucleic acidencodes a therapeutic protein that can reduce, prevent, and/or eliminatethe growth and/or survival of a cell, including the target cell.

Within certain aspects of these embodiments, the transcriptionalpromoter is activated in a target cell that is associated with adisease, condition, or age but is not activated in a normal mammaliancell that is not associated with the disease, condition, or aging.Target cell-specific transcriptional activation is achieved by theaction of one or more factors that are produced in the target cell butnot produced in a normal mammalian cell, including a normal human cell,such as normal skeletal myoblasts, normal adipose cells, normal cells ofthe eye, normal brain cells, normal liver cells, normal colon cells,normal lung cells, normal pancreas cells, and/or normal heart cells,which normal cells are not associated with the disease, condition, oraging.

Within other aspects of these embodiments, the target cell can be amammalian cell or a bacterial cell. Target mammalian cells can includehuman cells such as senescent cells, cancer cells, precancerous cells,dysplastic cells, and cells that are infected with an infectious agent.

In certain aspects of these embodiments wherein the human target cell isa senescent cell, the transcriptional promoter can include atranscriptional promoter, such as the p16INK4a/CDKN2A transcriptionalpromoter, which is responsive to activation by transcription factorssuch as SP1, ETS1, and/or ETS2. In other aspects of these embodimentswherein the human target cell is a senescent cell, the transcriptionalpromoter can include a transcriptional promoter, such as the p21/CDKN1Atranscriptional promoter, which is responsive to p53/TP53.

In a target cell, such as a senescent cell, transcriptional promotersinduce the expression of a nucleic acid that encodes a therapeuticprotein such as, for example, Casp3, Casp8, Casp9, BAX, DFF40, HSV-TK,and cytosine deaminase as well as inducible and self-activating variantsof Casp3, Casp8, Casp9, BAX, DFF40, HSV-TK, and cytosine deaminase whichtherapeutic protein reduces, prevents, and/or eliminates the growthand/or survival of the senescent cell, such as, for example, by inducingcell death in the senescent cell via a cellular process includingapoptosis. Other therapeutic proteins may be employed that reduce,prevent, and/or eliminate the growth and/or survival of a senescent cellby, for example, inducing cell death via a cellular process includingnecrosis/necroptosis, autophagic cell death, endoplasmicreticulum-stress associated cytotoxicity, mitotic catastrophe,paraptosis, pyroptosis, pyronecrosis, and entosifs.

In other aspects of these embodiments wherein the human target cell is acancer cell, such as a brain cancer cell, a prostate cancer cell, a lungcancer cell, a colorectal cancer cell, a breast cancer cell, a livercancer cell, a hematologic cancer cell, and a bone cancer cell, thetranscriptional promoter can include the p21^(cip1/waf1) promoter, thep27^(kip1) promoter, the p57^(kip2) promoter, the TdT promoter, theRag-1 promoter, the B29 promoter, the Blk promoter, the CD19 promoter,the BLNK promoter, and/or the λ5 promoter, which transcriptionalpromoter is responsive to activation by one or more transcriptionfactors such as an EBF3, O/E-1, Pax-5, E2A, p53, VP16, MLL, HSF1,NF-IL6, NFAT1, AP-1, AP-2, HOX, E2F3, and/or NF-κB transcription factor,and which transcriptional activation induces the expression of a nucleicacid that encodes a therapeutic protein such as, for example, Casp3,Casp8, Casp9, BAX, DFF40, HSV-TK, and cytosine deaminase as well asinducible and self-activating variants of Casp3, Casp8, Casp9, BAX,DFF40, HSV-TK, and cytosine deaminase which therapeutic protein reduces,prevents, and/or eliminates the growth and/or survival of the senescentcell, such as, for example, by inducing cell death in the senescent cellvia a cellular process including apoptosis. Other therapeutic proteinsmay be employed that reduce, prevent, and/or eliminate the growth and/orsurvival of a senescent cell by, for example, inducing cell death via acellular process including necrosis/necroptosis, autophagic cell death,endoplasmic reticulum-stress associated cytotoxicity, mitoticcatastrophe, paraptosis, pyroptosis, pyronecrosis, and entosifs.

In still further aspects of these embodiments wherein the target cell isa human cell that is infected with an infectious agent, such as a virus,including, for example, a herpes virus, a polio virus, a hepatitisvirus, a retrovirus virus, an influenza virus, and a rhino virus, or thetarget cell is a bacterial cell, the transcriptional promoter can beactivated by a factor that is expressed by the infectious agent orbacterial cell, which transcriptional activation induces the expressionof a nucleic acid that encodes a therapeutic protein such as, forexample, Casp3, Casp8, Casp9, BAX, DFF40, HSV-TK, and cytosine deaminaseas well as inducible and self-activating variants of Casp3, Casp8,Casp9, BAX, DFF40, HSV-TK, and cytosine deaminase which therapeuticprotein reduces, prevents, and/or eliminates the growth and/or survivalof the senescent cell, such as, for example, by inducing cell death inthe senescent cell via a cellular process including apoptosis. Othertherapeutic proteins may be employed that reduce, prevent, and/oreliminate the growth and/or survival of a senescent cell by, forexample, inducing cell death via a cellular process includingnecrosis/necroptosis, autophagic cell death, endoplasmicreticulum-stress associated cytotoxicity, mitotic catastrophe,paraptosis, pyroptosis, pyronecrosis, and entosifs.

Within other embodiments, the present disclosure provides systems forthe targeted production of a therapeutic protein within a target cell.These systems comprise a vector that is capable of delivering a nucleicacid to a cell, including a target cell as well as a non-target cell,wherein the vector comprises an expression construct for the targetedproduction of a therapeutic protein within a target cell (e.g., a cellthat is associated with age, disease, or other condition) but not withina non-target cell, wherein the expression construct comprises atranscriptional promoter that is activated in response to one or morefactors each of which is produced within said target cell; and a nucleicacid that is operably linked to and under regulatory control of thetranscriptional promoter, wherein the nucleic acid encodes a therapeuticprotein that can reduce, prevent, and/or eliminate the growth and/orsurvival of a cell in which it is produced, including a target cell.

Within certain aspects of these embodiments, formulations and systemsinclude lipid nanoparticle (LNP) formulations and systems wherein an LPNencapsulates a polynucleotide construct (e.g., a plasmid DNA) comprisinga coding region for a pro-apoptotic protein, such as a caspase protein,and wherein the coding region is under the regulatory control of atarget cell-specific transcriptional promoter, such as a senescentcell-specific transcriptional promoter or a cancer cell-specifictranscriptional promoter. Exemplary cell-specific transcriptionalpromoters include p16, p22, p53. Exemplary coding regions forpro-apoptotic proteins include coding regions for Casp3, Casp8, Casp9,BAX, DFF40, HSV-TK, and cytosine deaminase proteins. Pro-apoptoticproteins include inducible Casp3, Casp8, Casp9, BAX, DFF40, HSV-TK, andcytosine deaminase proteins and self-activating Casp3, Casp8, Casp9,BAX, DFF40, HSV-TK, and cytosine deaminase proteins, which areexemplified herein by an inducible Caspase 9 (iCasp9) or aself-activating Caspase 9 (saCasp9).

Inducible pro-apoptotic proteins, including iCasp9 proteins, can includea dimerization domain, such as an FKBP or FK506 binding protein domain,that binds to a chemical inducer of dimerization (CID), such as AP1903or AP20187. Clackson, Proc Natl Acad Sci USA. 95:10437-10442 (1998).Inducible Caspase 9 (iCasp9; Ariad, Erie, PA) may be activated in thepresence of AP1903. U.S. Pat. No. 5,869,337 and Straathof, Blood105:4247-4254 (2005). Exemplary human genes encoding FKBP domainsinclude AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FHBP5, FKBP6, FKBP7,FKBP8, FKBP8, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, FKBP52, andL00541473.

Within other aspects of these embodiments, lipid nanoparticles (LNP) arefusogenic lipid nanoparticles, such as fusogenic lipid nanoparticlescomprising a fusogenic protein, such as a fusogenic p14 FAST fusionprotein from reptilian reovirus to catalyze lipid mixing between the LNPand target cell plasma membrane. Suitable fusogenic proteins aredescribed in PCT Patent Publication Nos. WO2012/040825A1 andWO2002/044206A2, Lau, Biophys. J. 86:272 (2004), Nesbitt, Master ofScience Thesis (2012), Zijlstra, AACR (2017), Mrlouah, PAACRAM77(13Supp1):Abst 5143 (2017), Krabbe, Cancers 10:216 (2018),Sanchez-Garcia, ChemComm 53:4565 (2017), Clancy, J Virology 83(7):2941(2009), Sudo, J Control Release 255:1 (2017), Wong, Cancer Gene Therapy23:355 (2016), and Corcoran, JBC 281(42):31778 (2006) and areexemplified by the P14 and P14e15 proteins having the amino acidsequences presented in Table 1.

TABLE 1 Fusogenic Protein Sequences P14MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVIAG SEQ ID NO: 16LVALLTFLAFGFWLFKYLQKRRERRRQLTEFQKRYLRNSYRLSEIQRPISQHEYEDPYEPPSRRKPPPPPYSTYVNIDNVSAI* P14e15MGSGPSNFVNHAPGEAIVTGLEKGADKVAGTISHTIWEVIAG SEQ ID NO: 17LVALLTFLAFGFWLFKYLQWYNRKSKNKKRKEQIREQIELGLLSYGAGVASLPLLNVIAHNPGSVISATPIYKGPCTGVPNSRLLQITSGTAEENTRILNHDGRNPDGSINV*

Contacting a cell expressing an iCasp9 protein with a CID facilitatesthe dimerization of the iCasp9 protein, which triggers apoptosis in atarget cell. AP1903 has been used in humans multiple times, itsintravenous safety has been confirmed, and its pharmacokineticsdetermined. Iuliucci, J Clin Pharmacol 41(8):870-9 (2001) and Di Stasi,N Engl J Med 365:1673-83 (2011). iCasp9+AP1903 were used successfully inhumans to treat GvHD after allogeneic T cell transplant. Di Stasi, NEngl J Med 365:1673-83 (2011).

Within certain embodiments, a polynucleotide encoding a self-activatingcaspase, such as a self-activating Caspase 9 (saCasp9), may be employedwherein expression of the caspase polynucleotide is under the regulatorycontrol of a factor that is active in a target cell population, such asa senescent cell population or a cancer cell population. Self-activatingcaspases activate in the absence of a chemical inducer of dimerization(CID). Cells expressing self-activating caspases, such as saCasp9,apoptose almost immediately. It will be appreciated by those of skill inthe art that such self-activating caspases may be advantageouslyemployed for the induction of apoptosis in a rapidly dividing cell, suchas a rapidly dividing tumor cell, where an inducible caspase proteinwould be diluted out before administration of a CID. Moreover, becausecell death with a self-activating caspase occurs over a longer period oftime as compared to an inducible caspase, the risk of tumor lysissyndrome is reduced with a self-activating caspase.

Formulations comprising a plasmid DNA encapsulated with a LNPformulation are non-toxic and non-immunogenic in animals at doses of >15mg/kg and exhibit an efficiency in excess of 80×greater than thatachievable with neutral lipid formulations and 2-5×greater than thatachievable with cationic lipid formulations. LNP cargo is depositeddirectly into the cytoplasm thereby bypassing the endocytic pathway.

Within further aspect of these embodiments, the system further comprisesone or more safety features that permit additional control over theexpression of the nucleic acid within the expression construct or thefunctionality of a therapeutic protein encoded by the nucleic acid suchas, for example, by requiring the contacting of a target cell with achemical or biological compound that, in addition to the intracellularfactor that promotes transcriptional activation of the promoter withinthe expression construct or promotes the functionality of thetherapeutic protein, such as by promoting the dimerization of as well asinducible variants of Casp3, Casp8, Casp9, BAX, DFF40, HSV-TK, andcytosine deaminase.

A further safety element that may be employed in the expressionconstructs and systems of the present disclosure includes atamoxifen-inducible Cre construct using Life Technologies GatewayCloning Vector System employing a pDEST26 plasmid for mammalianexpression. For example, a fusion protein of Cre and estrogen receptorcan be constitutively expressed and induced upon the addition oftamoxifen, which permits activated Cre to re-orient the transcriptionalpromoter, thereby expressing the therapeutic protein.

Within yet other aspects of these embodiments, the system may furthercomprise a nucleic acid that encodes a detectable marker, such as abioluminescent marker, thereby allowing the identification of cells thatexpress the therapeutic protein and, in the case of an inducibletherapeutic protein such as an inducible Casp3, Casp8, Casp9, will bekilled by the administration of a compound that promotes activity of thetherapeutic protein, such as by inducing the dimerization of aninducible Casp3, Casp8, Casp9.

Within further embodiments, the present disclosure provides methods forreducing, preventing, and/or eliminating the growth of a target cell,which methods comprise contacting a target cell with a system for thetargeted production of a therapeutic protein within a target cell,wherein the system comprises a vector that is capable of delivering anucleic acid to a cell, wherein the vector comprises an expressionconstruct for the targeted production of a therapeutic protein within atarget cell (e.g., a cell that is associated with age, disease, or othercondition) but not within a non-target cell, wherein the expressionconstruct comprises: (a) a transcriptional promoter that is activated inresponse to one or more factors each of which factors is produced withina target cell and (b) a nucleic acid that is operably linked to andunder regulatory control of the transcriptional promoter, wherein thenucleic acid encodes a therapeutic protein that is produced uponexpression of the nucleic acid and wherein production of the therapeuticprotein in the target cell (i.e., the cell that is associated with age,disease, or other condition) reduces, prevents, and/or eliminates growthand/or survival of the target cell.

Within still further embodiments, the present disclosure providesmethods for the treatment of an aging human or a human that is afflictedwith a disease or another condition, wherein the aging, disease, orother condition is associated with a target cell within the human, themethods comprising administering to the human a system for the targetedproduction of a therapeutic protein within a target cell, wherein thesystem comprises a vector that is capable of delivering a nucleic acidto a cell, wherein the vector comprises an expression construct for thetargeted production of a therapeutic protein within a target cell (e.g.,a cell that is associated with age, disease, or other condition) but notwithin a non-target cell, wherein the expression construct comprises:(a) a transcriptional promoter that is activated in response to one ormore factors each of which factors is produced within a target cell and(b) a nucleic acid that is operably linked to and under regulatorycontrol of the transcriptional promoter, wherein the nucleic acidencodes a therapeutic protein that is produced upon expression of thenucleic acid and wherein production of the therapeutic protein in thetarget cell (i.e., the cell that is associated with age, disease, orother condition) reduces, prevents, and/or eliminates growth and/orsurvival of the target cell thereby slowing aging in the human and/orslowing, reversing, and/or eliminating the disease or condition in thehuman.

Within further embodiments, the present disclosure provides lipidnanoparticle (LNP) formulation for the targeted production of atherapeutic protein within a target cell, which LNP formulationcomprise: (a) a lipid nanoparticle vector for the non-specific deliveryof a nucleic acid to mammalian cells, which mammalian cells include bothtarget cells or non-target cells, wherein said lipid nanoparticleincludes one or more lipid(s) and one or more fusogenic protein(s), and(b) an expression construct for the preferential production of atherapeutic protein within a target cell.

LNP formulations according to certain aspects of these embodimentsinclude one or more lipid(s) at a concentration ranging from 1 mM to 100mM, or from 5 mM to 50 mM, or from 10 mM to 30 mM, or from 15 mM to 25mM. LNP formulations exemplified herein include one or more lipid(s) ata concentration of about 20 mM.

Within certain illustrative LNP formulations, one or more lipid(s) isselected from 1,2-dioleoyl-3-dimethyl ammonium-propane (DODAP), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Cholesterol, and1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG). LNPformulations may two or more lipids selected from the group consistingof DODAP, DOTAP, DOPE, Cholesterol, and DMG-PEG.

Exemplified herein are LNP formulations including DODAP, DOTAP, DOPE,Cholesterol, and DMG-PEG at a molar ratio of 35-55 mole % DODAP:10-20mole % DOTAP:22.5-37.5 mole % DOPE:4-8 mole % Cholesterol:3-5 mole %DMG-PEG; or at a molar ratio of about 45 mole % DODAP:about 15 mole %DOTAP:about 30 mole % DOPE:about 6 mole % Cholesterol:about 4 mole %DMG-PEG. Within certain aspects, the LNP formulations include DODAP,DOTAP, DOPE, Cholesterol, and DMG-PEG at a molar ratio of 45 mole %DODAP:15 mole % DOTAP:30 mole % DOPE:6 mole % Cholesterol:4 mole %DMG-PEG.

LNP formulations according to other aspects of these embodiments includeone or more fusogenic protein(s) at a concentration ranging from 0.5 μMto 20 or from 1 μM to 10 μM, or from 3 μM to 4 μM. Exemplified hereinare LNP formulations wherein fusogenic protein(s) are present at aconcentration of 3.5 μM. Exemplary, suitable fusogenic protein(s)include the p14 fusogenic protein (SEQ ID NO: 16) and a the p14e15fusogenic protein (SEQ ID NO: 17).

Within additional aspects of these embodiments, LNP formulations includeexpression constructs comprising (i) a transcriptional promoter that isactivated in response to one or more factors that are preferentiallyproduced within said target cells as compared to said non-target cellsand (ii) a nucleic acid that is operably linked to and under regulatorycontrol of said transcriptional promoter, wherein said nucleic acidencodes a therapeutic protein that can reduce, prevent, and/or eliminatethe growth and/or survival of mammalian cells, including both targetcells and non-target cells and wherein said therapeutic protein isproduced within said target cells but is not produced in said non-targetcells.

Exemplified herein are LNP formulations including expression constructsat a concentration ranging from 20 μg/mL to 1.5 mg/mL, of from 100 μg/mLto 500 μg/mL, or at a concentration of 250 μg/mL.

A suitable exemplary LNP formulation includes the following: for each 1mL of LNP, the lipid concentration is 20 mM, the DNA content is 250 μg,and the fusogenic protein (e.g., p14 or p14e15) is at 3.5 μM wherein thelipid formulation comprises DODAP:DOTAP:DOPE:Cholesterol:DMG-PEG at amole % ratio of 45:15:30:6:4, respectively.

Within still further aspects of these embodiments, LNP formulationsinclude expression constructs having a transcriptional promoter selectedfrom a p16 transcriptional promoter, a p21 transcriptional promoter, anda p53 transcriptional promoter, and include transcriptional promotersthat are responsive to a factor selected from SP1, ETS1, ETS2, andp53/TP53. Exemplified herein are LNP formulations wherein saidtranscriptional promoter is a p16INK4a/CDKN2A transcriptional promoteror a p21/CDKN1A transcriptional promoter.

Within related aspects of these embodiments, LNP formulations includeexpression constructs having a transcriptional promoter that isresponsive to a factor selected from EBF3, O/E-1, Pax-5, E2A, p53, VP16,MLL, HSF1, NF-IL6, NFAT1, AP-1, AP-2, HOX, E2F3, and/or NF-κB.Exemplified herein are LNP formulations wherein said transcriptionalpromoter is a p21^(cip1/waf1) promoter, the p27^(kip1) promoter, thep57^(kip2) promoter, the TdT promoter, the Rag-1 promoter, the B29promoter, the Blk promoter, the CD19 promoter, the BLNK promoter, andthe λ5 promoter.

Within other related aspects of these embodiments, LNP formulationsinclude expression constructs that include a nucleic acid that encodes atherapeutic protein, such as a therapeutic protein selected from acaspase (Casp), an inducible caspase (iCasp), a self-activating caspase(saCasp), BAX, DFF40, HSV-TK, and cytosine deaminase. Exemplified hereinare LNP formulations that include expression constructs having a nucleicacid that encodes a Casp9, including, for example, an inducible Casp9(iCasp9) or a self-activating Casp9 (saCasp9).

Other embodiments of the present disclosure provide methods forreducing, preventing, and/or eliminating the growth of a target cell,which comprise contacting a target cell with an LNP formulation having(a) a lipid nanoparticle vector for the non-specific delivery of anucleic acid to mammalian cells, which mammalian cells include bothtarget cells or non-target cells, wherein said lipid nanoparticleincludes one or more lipid(s) and one or more fusogenic protein(s), and(b) an expression construct for the preferential production of atherapeutic protein within a target cell.

Within certain aspects of these embodiments the methods employ LNPformulations comprising (i) a transcriptional promoter that is activatedin response to one or more factors that are preferentially producedwithin target cells as compared to non-target cells and (ii) a nucleicacid that is operably linked to and under regulatory control of thetranscriptional promoter, wherein the nucleic acid encodes a therapeuticprotein that can reduce, prevent, and/or eliminate the growth and/orsurvival of mammalian cells, including both target cells and non-targetcells and wherein said therapeutic protein is produced within the targetcells but is not produced in the non-target cells.

Other embodiments of the present disclosure provide methods for thetreatment of a disease or condition in a patient, including a humanpatient, having a target cell, wherein the method comprisesadministering to the patient an LNP formulation having (a) a lipidnanoparticle vector for the non-specific delivery of a nucleic acid tomammalian cells, wherein the mammalian cells include both target cellsor non-target cells, and wherein the lipid nanoparticle includes one ormore lipid(s) and one or more fusogenic protein(s) and (b) an expressionconstruct for the preferential production of a therapeutic proteinwithin a target cell.

Within certain aspects of these embodiments the methods employ LNPformulations comprising (i) a transcriptional promoter that is activatedin response to one or more factors that are preferentially producedwithin target cells as compared to non-target cells and (ii) a nucleicacid that is operably linked to and under regulatory control of thetranscriptional promoter, wherein the nucleic acid encodes a therapeuticprotein that can reduce, prevent, and/or eliminate the growth and/orsurvival of mammalian cells, including both target cells and non-targetcells and wherein said therapeutic protein is produced within the targetcells but is not produced in the non-target cells.

These and other related aspects of the present disclosure will be betterunderstood in light of the following drawings and detailed description,which exemplify certain aspects of the various embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of conventional and fusogenicliposomes, including stealth fusogenic liposomes, including lipidnanoparticles employing Innovascreen's Fusogenix™ Platform according tocertain aspects of the present disclosure. Shown are Fusogenix™ lipidnanoparticles utilizing a p14 FAST fusion protein from reptilianreovirus and including a plasmid vector encoding an inducible Caspase 9(iCasp9) under a promoter that is active in a target cell population,such as a senescent target cell population or a cancer target cellpopulation. Exemplified in this diagram are Casp9 fusion peptides thatare activated via a small molecule dimerizer such as AP1903.

FIG. 2 is a diagrammatic representation of the liposomal delivery to thecytoplasm of a target cell, according to certain aspects of the presentdisclosure. Shown are Fusogenix™ lipid nanoparticles (LNPs) that areconfigured for the delivery of nucleic acids, such as those encoding apro-apoptotic protein, such as Caspase 9, under the regulatory controlof a target cell-specific transcriptional promoter, such as a targetsenescent cell encoding p16 or a target cancer cell encoding p53.Exemplified are Fusogenix™ lipid nanoparticles comprising a p14 FASTprotein to catalyze the rapid lipid mixing between the lipidnanoparticle (LNP) and the target cell plasma membrane. Such Fusogenix™lipid nanoparticles (i) deliver the cargo nucleic acids directly intothe cytoplasm thereby bypassing the endocytic pathway, (ii) arenon-toxic (i.e., non-immunogenic) in animals at doses of ≥15 mg/kg,(iii) are 80× more efficient than neutral lipid formulations, (iv) are2-5× more efficient than cationic lipid formulations, and (iv) aremanufacturable at scale.

FIG. 3 is a table comparing the reported maximum tolerated dose (MTD)for clinical stage lipid-based in vivo delivery technologies. The MTDof >15 mg/kg for fusogenic lipid nanoparticles of the present disclosurewas estimated from rat toxicity data.

FIG. 4A is a diagrammatic representation of the induction of aninducible Caspase 9 homodimer (iCasp9), which iCasp9 is a fusion proteincomprising a drug-binding domain for binding to a chemical inducer ofdimerization (CID) and an active portion of Caspase 9. A CID, asexemplified by CIDs designated AP1903 and AP20187, binds to thedrug-binding domain of the iCasp9 fusion protein to dimerize and,thereby, activate iCasp9, which results in the intracellular activationof pro-apoptotic molecules and the induction of apoptosis within atarget cell.

FIG. 4B is a diagrammatic representation of an exemplary apoptosomeaccording to certain aspects of the present disclosure.

FIG. 5 depicts the chemical structure of an exemplary chemical inducerof dimerization (CID), which is a homodimerizer designated herein asAP1903 (APExBIO, Houston, TX) that may be employed in variousembodiments of the present disclosure for inducing the activity of aninducible pro-apoptotic protein, such as an inducible caspase protein(e.g., iCasp9).

FIG. 6 depicts the chemical structure of an exemplary chemical inducerof dimerization (CID), which is a homodimerizer designated herein asAP20187 (APExBIO, Houston, TX) that may be employed in variousembodiments of the present disclosure for inducing the activity of aninducible pro-apoptotic protein, such as an inducible caspase protein(e.g., iCasp9).

FIGS. 7A-7B present data obtained in mice that were administeredintravenously Fusogenix lipid nanoparticles labeled with ⁶⁴Cu-NOTA[1,4,7-triazacyclononane-1,4,7-triacetic acid]. See, Fournier, EJNMMIResearch 2:8 (2012). ⁶⁴Cu was detected via positron emission tomography(PET). FIG. 7A presents PET data obtained from a mouse to which⁶⁴Cu-NOTA-liposomes without protein were administered. FIG. 7B presentsPET data obtained from a mouse to which ⁶⁴Cu-NOTA-liposome-p14 wereadministered.

FIG. 8 is a bar graph of data obtained with Fusogenix lipidnanoparticles comparing SUV_(mean, 24 h) for ⁶⁴Cu-NOTA-liposomes withoutprotein and ⁶⁴Cu-NOTA-liposome-p14. The data presented in FIGS. 7 and 8demonstrate a 50% increase in gene/siRNA delivery to prostate tumors ascompared to a competing formulation.

FIG. 9 is a bar graph of the biodistribution of labelled pegylatedliposomes in nude mice expressed after 24 hours as discussed in Example1.

FIGS. 10 and 11 are graphs of optical density at 405 nm as a function ofconcentration (μg/ml; FIG. 10 ) and of anti-p14 and anti-LNP antibodyresponses (FIG. 11 ), which demonstrate the safety and tolerability ofexemplary fusogenic lipid nanoparticles utilizing a reptilian reovirusp14 FAST fusion protein (Fusogenix™). As shown, virtually no antibodyresponse was observed in immune competent mice (with and withoutadjuvant).

FIGS. 12 and 13 are bar graphs of data from in vitro anti-p14 andanti-LNP antibody neutralization assays showing that lipid nanoparticleformulations according to the present disclosure are non-reactive withC4d (FIG. 12 ) and less reactive with iC3b (FIG. 13 ) as compared toDoxil in 8 out of 10 human samples tested for Complementactivation-related psuedoallergy (CARPA) using C4d and iC3b complementELISA assays as described in Szebeni, Mol Immunol 61(2):163-73 (2014).

FIG. 14 is a restriction map of the plasmid vector pVAX1™ which isemployed in certain aspects of the expression constructs, systems,formulations, and methods of the present disclosure for the targetcell-specific production of a therapeutic protein, such as apro-apoptotic protein, including a caspase protein, such as Caspase 9,as well as inducible and self-activating variants of a pro-apoptoticprotein, including inducible and self-activating variants of caspaseproteins, such as inducible Caspase 9 (iCasp9) and self-activatingCaspase 9 (saCasp9). In certain embodiments, expression constructs andformulations may additionally include a safety element, such as atamoxifen-inducible Cre construct (e.g., Life Technologies GatewayCloning Vector System). A fusion protein of Cre and estrogen receptor isconstitutively expressed and induced upon the addition of tamoxifen,which permits activated Cre to re-orient the p16-promoter, therebyexpressing caspase 9 or inducible/self-activating variant thereof pVAX1is commercially available from ThermoFisher Scientific (Waltham, MA).

FIG. 15 is a diagrammatic representation of an exemplary p16-targetingconstruct for the target cell-specific expression of an inducibleCaspase 9 (iCasp9) or a self-activating Caspase 9 (saCasp9) protein intarget cells expressing p16, such as target cells that are associatedwith aging and/or senescence, which p16-targeting construct comprises ap16s transcriptional promoter in operable connection to iCasp9 orsaCasp9. An exemplary p16 transcriptional promoter is described in Bakeret al., Nature 479(7372):232-67 (2011)).

FIG. 16 is a restriction map of the plasmid vector pVAX1-16s-iCasp9-MX(SEQ ID NO: 6), which comprises an exemplary p16-targeting construct forthe target cell-specific expression of an inducible Caspase 9 (iCasp9)protein in target cells expressing p16, such as target cells that areassociated with aging and/or senescence, which p16-targeting constructcomprises a p16s transcriptional promoter in operable connection toiCasp9.

FIG. 17 is a plasmid map of the vector p10-p16-iCasp9 (SEQ ID NO: 12),which comprises an exemplary p16-targeting construct for the targetcell-specific expression of an inducible Caspase 9 (iCasp9) protein intarget cells expressing p16, such as target cells that are associatedwith aging and/or senescence, which p16-targeting construct comprises ap16e transcriptional promoter in operable connection to iCasp9.

FIG. 18 is a plasmid map of the vector p10-p16-saCasp9 (SEQ ID NO: 13),which comprises an exemplary p16-targeting construct for the targetcell-specific expression of an self-activating Caspase 9 (saCasp9)protein in target cells expressing p16, such as target cells that areassociated with aging and/or senescence, which p16-targeting constructcomprises a p16e transcriptional promoter in operable connection tosaCasp9.

FIG. 19 is a diagrammatic representation of the in vivo administrationof an exemplary p16-targeting construct in an mouse model system foraging, wherein the aging mouse model exhibits a senescent cell burden(as defined by the presence of p16⁺ cells) and secretion of factorsassociated with a senescence-associated secretory phenotype (SASP; vanDeursen, Nature 509(7501):439-446 (2014)). A formulation comprising avector and an expression construct, such as a lipid nanoparticle (LNP)vector, e.g., a fusogenic LNP comprising a fusogenic protein such as p14FAST, encompassing a p16-Casp9 expression construct, e.g.,pVAX1-16s-iCasp9, p10-p16e-iCasp9, p10-p16e-saCasp9, or variant thereofexpressing luciferase (for visualization), is administered in vivo to anaged mouse via injection into a tail vein and the LNP+expressionconstruct transfects target and non-target cells without specificity.Upon subsequent in vivo administration of a chemical inducer ofdimerization (CID), such as AP20187, p16+ target cells (e.g., senescentcells) expressing an iCasp9 protein undergo apoptosis, resulting in areduction is SASP levels, while p16− cells remain viable.

FIGS. 20A-20C are photomicrographs of the histiological staining ofsenescent-associated β-gal in kidney cells from an in vivo aged mousemodel either untreated (FIG. 20A) or treated (low dose—FIG. 20B and highdose—FIG. 20C) following the in vivo administration (16 animals at 80weeks of age) of a formulation comprising a vector and an expressionconstruct, such as a lipid nanoparticle (LNP) vector, e.g., a fusogenicLNP comprising a fusogenic protein such as p14 FAST, encompassing ap16-Casp9 expression construct, e.g., pVAX1-16s-iCasp9, p10-p16e-iCasp9,p10-p16e-saCasp9, or variant thereof, is administered in vivo to an agedmouse and kidney cells stained for β-gal. These data demonstrated adose-dependent reduction of p16+ senescent kidney cells (FIG. 20D).

FIGS. 20E-20G are photomicrographs of the histiological staining ofsenescent-associated β-gal in seminal vesicle cells from an in vivo agedmouse model either untreated (FIG. 20E) or treated (low dose—FIG. 20Fand high dose—FIG. 20G) following the in vivo administration (16 animalsat 80 weeks of age) of a formulation comprising a vector and anexpression construct, such as a lipid nanoparticle (LNP) vector, e.g., afusogenic LNP comprising a fusogenic protein such as p14 FAST,encompassing a p16-iCasp9 expression construct, e.g., pVAX1-16s-iCasp9,p10-p16e-iCasp9, p10-p16e-saCasp9, or variant thereof, is administeredin vivo to an aged mouse and seminal vesicle cells stained for β-gal.These data demonstrated a dose-dependent reduction of p16+ senescentseminal vesicle cells (FIG. 2011 ).

FIG. 21 is a bar graph demonstrating the dose-dependent targeting ofp16+ kidney cells in naturally aged mice following the in vivoadministration of a fusogenic lipid nanoparticle (LNP) formulationcomprising a pVAX1-p16 expression construct. Kidney cells were subjectedto a qRT-PCR reaction to detect p16^(Ink4a) transcripts. Relativeexpression was calculated using 2ΔΔCt (Livak, Methods 25:402-408(2001)).

FIG. 22 is a bar graph demonstrating the dose-dependent targeting ofp16+ spleen cells in naturally aged mice following the in vivoadministration of a fusogenic lipid nanoparticle (LNP) formulationcomprising a pVAX1-p16 expression construct. Spleen cells were subjectedto a qRT-PCR reaction to detect p16^(Ink4a) transcripts. Relativeexpression was calculated using 2ΔΔCt (Livak, Methods 25:402-408(2001)).

FIG. 23 is a bar graph demonstrating the dose-dependent targeting ofp16+ seminal vesicle cells in naturally aged mice following the in vivoadministration of a fusogenic lipid nanoparticle (LNP) formulationcomprising a pVAX1-p16 expression construct. Seminal vesicle cells weresubjected to a qRT-PCR reaction to detect p16^(Ink4a) transcripts.Relative expression was calculated using 2ΔΔCt (Livak, Methods25:402-408 (2001)).

FIG. 24 is a bar graph demonstrating the dose-dependent targeting ofp16+ inguinal fat cells in naturally aged mice following the in vivoadministration of a fusogenic lipid nanoparticle (LNP) formulationcomprising a pVAX1-p16 expression construct. Inguinal fat cells weresubjected to a qRT-PCR reaction to detect p16^(Ink4a) transcripts.Relative expression was calculated using 2ΔΔCt (Livak, Methods25:402-408 (2001)).

FIG. 25 is a bar graph demonstrating the dose-dependent targeting ofp16+ lung cells in naturally aged mice following the in vivoadministration of a fusogenic lipid nanoparticle (LNP) formulationcomprising a pVAX1-p16 expression construct. Lung cells were subjectedto a qRT-PCR reaction to detect p16^(Ink4a) transcripts. Relativeexpression was calculated using 2ΔΔCt (Livak, Methods 25:402-408(2001)).

FIG. 26 is a bar graph of data demonstrating the remediation ofchemotherapy-induced damage (as determined by the clearance of damagedcells (i.e., senescent cells) after treatment with doxorubicin).Senescence was induced in B6 mice with doxorubicin. Animals were treatedwith murine p53-iCasp9 and dimerizer or controls (dimerizer only and LNPonly) and sacrificed. Tissues were assayed for p53 expression viart-PCR.

FIG. 27 is a diagrammatic representation of an exemplary p53-targetingcassette for use in treatment of cancers (oncology) by the selectivekilling of tumor cells according certain embodiments of the presentdisclosure. The p53-targeting cassette comprises a p53 transcriptionalpromoter, which drives the expression an inducible caspase 9 protein(iCasp9) or a self-activating caspase 9 protein (saCasp9).

FIG. 28 is a restriction map of a plasmid (pVAX1-p53-iCasp9-MX; SEQ IDNO: 7) comprising a p53-targeting cassette as depicted in FIG. 27 .Expression of an iCasp9 nucleic acid encoding an inducible Casp9 proteinis regulated by the p53 transcriptional promoter.

FIG. 29 is a restriction map of a plasmid (pVAX1-p53-saCasp9; SEQ ID NO:8) comprising a p53-targeting cassette. Expression of a nucleic acidencoding a self-activating Caspase 9 (saCasp9) protein is regulated bythe p53 transcriptional promoter.

FIG. 30 is a restriction map of a plasmid (pVAX1-p53-iCasp9-OVA; SEQ IDNO: 11) comprising a p53-targeting cassette as depicted in FIG. 27 .Expression of a nucleic acid encoding an inducible Casp9 protein isregulated by the p53 transcriptional promoter.

FIG. 31 is a restriction map of a plasmid (pVAX1-p53-iCasp9-G-O; SEQ IDNO: 9) comprising a p53-targeting cassette as depicted in FIG. 27 .Expression of an iCasp9 nucleic acid encoding an inducible Casp9 proteinis regulated by the p53 transcriptional promoter.

FIG. 32 is a restriction map of a plasmid (pVAX1-p53-iCasp9-huCD40L; SEQID NO: 10) comprising a p53-targeting cassette as depicted in FIG. 27 .Expression of an iCasp9 nucleic acid encoding an inducible Casp9 proteinis regulated by the p53 transcriptional promoter. Additional targetingcassettes and plasmid constructs have been developed for advancedoncology applications, as disclosed herein, which constructs employnucleic acids encoding, for example, one or more immunostimulatorycytokines (such as huCD40L, as shown in FIG. 32 , as well as GMCSF andIL12) and/or one or more antigens (such as chicken ovalbumin (OVA), asshown in FIG. 30 , as well as Nt1, tetanus antigens, and influenzaantigens).

FIG. 33 is a map of a plasmid (p10-p53e-iCasp9; SEQ ID NO: 14)comprising a p53-targeting cassette as depicted in FIG. 27 . Expressionof an iCasp9 nucleic acid encoding an inducible Casp9 protein isregulated by the p53 transcriptional promoter. Additional targetingcassettes and plasmid constructs have been developed for advancedoncology applications, as disclosed herein, which constructs employnucleic acids encoding, for example, one or more immunostimulatorycytokines (such as huCD40L, as shown in FIG. 32 , as well as GMCSF andIL12) and/or one or more antigens (such as chicken ovalbumin (OVA), asshown in FIG. 30 , as well as Nt1, tetanus antigens, and influenzaantigens).

FIG. 34 is a map of a plasmid (p10-p53e-saCasp9; SEQ ID NO: 15)comprising a p53-targeting cassette as depicted in FIG. 27 . Expressionof an saCasp9 nucleic acid encoding a self-activating Casp9 protein isregulated by the p53 transcriptional promoter. Additional targetingcassettes and plasmid constructs have been developed for advancedoncology applications, as disclosed herein, which constructs employnucleic acids encoding, for example, one or more immunostimulatorycytokines (such as huCD40L, as shown in FIG. 32 , as well as GMCSF andIL12) and/or one or more antigens (such as chicken ovalbumin (OVA), asshown in FIG. 30 , as well as Nt1, tetanus antigens, and influenzaantigens).

FIG. 35 is a diagram showing the rationale for targeting p53+ tumorswith expression constructs comprising a p53 promoter in operablecombination with a pro-apoptotic protein, such as a caspase protein,e.g., a Caspase 9 protein. Cancer cells often mutate or delete it sothey can grow uncontrollably. However, even when the p53 gene ismutated, the transcription factors that bind to it are almost alwaysstill active.

FIG. 36 is a Western blot of iCasp 9 and Casp 9 protein levels obtainedwith p53-expressing cells (pVax-p53) and control cells (pcDNA3-GFP).Human prostate cancer PC-3 cells were treated with Fusogenix lipidnanoparticles carrying the pVax-p53-iCasp9-luc (luciferin) plasmid (inthe presence and absence of the homodimerizer AP201870) and assessed foriCasp9 expression. These data demonstrate that addition of the chemicalinducer of dimerization (CID; e.g., AP20187 and AP1903) abolishes theexpression of iCasp9 and luciferase in p53-expressing cells engineeredto express iCasp9 or luciferase.

FIGS. 37A-37D are microscopic images of human prostate cancer (LNCaP,DU145, PC-3) or normal epithelial (RWPE) cells treated with Fusogenixlipid nanoparticles carrying the pVax-p53-iCasp9-luc plasmid andassessed for iCasp9 expression by Western blot (data not shown) andluminescence assays 24 hours after exposure to EtOH (negative control,FIG. 37A and FIG. 37B) or AP1903 (FIG. 37C and FIG. 37D).

FIGS. 38-41 are bar graphs of data obtained with the p53-expressingcells presented in FIG. 37 . Human prostate cancer (LNCaP (FIG. 38 ),DU145 (FIG. 39 ), PC-3 (FIG. 40 )) or normal epithelial (RWPE (FIG. 41)) cells were treated with Fusogenix lipid nanoparticles carrying thepVax-p53-iCasp9-luc plasmid and assessed for iCasp9 expression byWestern blot and luminescence assays. These data demonstrate thataddition of the chemical inducer of dimerization (CID; e.g., AP20187 andAP1903) abolishes the expression of iCasp9 and luciferase inp53-expressing cells engineered to express iCasp9 or luciferase.

FIG. 42 is a bar graph of data from a luminescence assay of iCasp 9 andCasp 9 protein levels obtained with the p53-expressing cells presentedin FIG. 36 (pVax-p53) and control cells (pcDNA3-GFP). Human prostatecancer PC-3 cells were treated with Fusogenix lipid nanoparticlescarrying the pVax-p53-iCasp9-luc (luciferin) plasmid (in the presenceand absence of the homodimerizer AP20187) and assessed for iCasp9expression. These data demonstrate that addition of the chemical inducerof dimerization (CID; e.g., AP20187 and AP1903) abolishes the expressionof iCasp9 and luciferase in p53-expressing cells engineered to expressiCasp9 or luciferase.

FIGS. 43A, 43B, 44A, and 44B are flow cytometry apoptosis data (AnnexinV) from human prostate cancer PC-3 cells treated with pVax-p53 Fusogenixlipid nanoparticles (in the absence and presence of AP20187, FIGS. 43Aand 44A and 43B and 44B, respectively). The data presented in thesefigures demonstrates that suicide gene therapy selectively killsp53-expressing human prostate cancer cells in culture by inducingapoptosis (Luciferase-Annexin V flow cytometry).

FIG. 45 is a flow diagram depicting a pre-clinical oncology studyaccording to the present disclosure with 30×NSG mice implanted withhuman prostate tumor cells.

FIG. 46 is a graph of tumor volume (mm³) from the pre-clinical oncologystudy depicted in FIG. 33 in which NSG mice bearing a subcutaneous humanprostate PC-3 tumor was injected intratumorally (IT) with 100 μgFusogenix pVax-p53 formulation, followed 96 hours later by intravenous(IV) administration of 2 mg/kg of the homodimerizer AP20187.

FIGS. 47A-47C are photographs of tumors from the IT injection oncologystudy of FIG. 46 in which NSG mouse bearing a subcutaneous humanprostate PC-3 tumor was injected intratumorally with 100 μg FusogenixpVax-p53 formulation, followed 96 hours by 2 mg/kg AP20187 IV. FIG. 47Ashows tumor mass prior to administration of AP20187, FIG. 47B showstumor mass at 24 hours following administration of AP20187, and FIG. 47Cshows tumor mass at 96 hours following administration of AP20187.

FIG. 48 is a graph from the first of four NSG mice bearing subcutaneoushuman prostate cancer PC-3 tumors that were injected intravenously (IV)with 4×100 μg doses of Fusogenix pVax-p53 formulation, followed 24 hourslater by 2 mg/kg AP20187 IV.

FIG. 49 is a graph from the second of four NSG mice bearing subcutaneoushuman prostate cancer PC-3 tumors that were injected intravenously (IV)with 4×100 μg doses of Fusogenix pVax-p53 formulation, followed 24 hourslater by 2 mg/kg AP20187 IV.

FIG. 50 is a graph from the third of four NSG mice bearing subcutaneoushuman prostate cancer PC-3 tumors that were injected intravenously (IV)with 4×100 μg doses of Fusogenix pVax-p53 formulation, followed 24 hourslater by 2 mg/kg AP20187 IV.

FIG. 51 is a graph from the fourth of four NSG mice bearing subcutaneoushuman prostate cancer PC-3 tumors that were injected intravenously (IV)with 4×100 μg doses of Fusogenix pVax-p53 formulation, followed 24 hourslater by 2 mg/kg AP20187 IV.

FIG. 52 is a graph showing the percentage change in tumor volume as afunction of time after in vivo administration of a chemical inducer ofdimerization (CID) in NSG mice (N=6 for all groups) bearing a prostatetumor that were treated with intravenous p14 LNP pVAX.

FIG. 53 is a survival curve showing the percent survival as a functionof time after in vivo administration of a chemical inducer ofdimerization (CID) in NSG mice (N=6 for all groups) bearing a prostatetumor that were treated with intravenous p14 LNP pVAX.

FIG. 54 is a graph of dose escalation data showing the percentage changein tumor volume as a function of time after in vivo administration of achemical inducer of dimerization (CID) in NOD-SCID mice (N=6 for allgroups) bearing a prostate tumor that were treated with 100 μg, 400 μg,and 1000 μg of intravenous p14 LNP pVAX. NOD-SCID mice were implantedsubcutaneously with 500,000 PC-3 cells and randomized into treatmentgroups when their tumors reached 200 mm³, (N=2 for all groups). Animalswere injected with their assigned dose of p53-iCasp9 LNP IV twicefollowed by 2 mg/kg dimerizer. Tumors were measured directly every 24hours.

FIG. 55 is a graph showing the suppression of metastatic tumor growthwith repeat treatment of a p53-iCasp9 LNP with or without a chemicalinducer of dimerization (CID). NOD-SCID mice were injected with 500,000PC-3M-luciferase cells on Day 0, LNP dosing was started on Day 22 with150 μg p53-iCasp9 LNP. Dimerizer doses started Day 24 at 2 mg/kg. Micewere imaged every 24-48 hours to detect whole animal luminescence.

FIGS. 56 and 57 are graphs showing the percentage change in tumor volume(FIG. 56 ) and percent survival (FIG. 57 ) as a function of time afterin vivo administration of a chemical inducer of dimerization (CID) inisogenic C57B6 mice implanted with B16 murine melanoma cells treatedwith LNPs containing a construct encoding iCasp9 and murine CD40L undercontrol of the murine p53 promoter. Even though the rapid (10 hour)doubling time of the B16 cells made them largely refractory to theiCasp9-induced apoptosis, they still secreted enough CD40L toeffectively halt the tumor's growth. A construct encoding GMCSF+OVAantigen was also tested and determined to be more effective than iCasp9alone, but less effective than the CD40L version. N=3 for both groups.

FIGS. 58A-58D and FIG. 59 are photographs and a bar graph, respectively,of a B15F10 lung metastasis model data in which 100 μg of a control LNP(FIGS. 58A and 58B) or a p53-iCasp9 LNP (FIGS. 58C and 58D) wasadministered intravenously at days 3, 6, 9, and 12 following theintravenous injection of 75,000 B16F10 cells. At days 5, 8, 11, and 13,a chemical inducer of dimerization (CID) was administeredintraperitoneally. Animals were sacrificed at day 14 and lung metastaseswere quantified.

FIG. 60 and FIG. 61 are DEXA scans, which were performed monthly, afterin vivo administration of LNP formulations targeting p16, p53, or thecombination (p16+p53) (N=10 for all groups). Mice were treated monthlystarting at 728 days (104 weeks) of age.

FIG. 62 and FIG. 63 are graphs showing the change in bone density inmale (FIG. 62 ) and female (FIG. 63 ) naturally aged mice after in vivoadministration of LNP formulations targeting p16, p53, or thecombination (p16+p53) (N=10 for all groups). Mice were treated monthlystarting at 728 days (104 weeks) of age (arrows). At 896 days (128weeks), the increase in bone density benefit for treated mice isapparent in the male mice.

FIG. 64 is a survival curve showing the percent survival as a functionof time after in vivo administration of LNP formulations targeting p16,p53, or the combination (p16+p53) (N=10 for all groups). Mice weretreated monthly starting at 728 days (104 weeks) of age (arrows). At 931days (133 weeks), the survival benefit for treated mice is apparent(>50% survival difference between combination treatment and control).

DETAILED DESCRIPTION

The present disclosure provides expression cassettes, systems, andmethods for the selective reduction, prevention, and/or elimination inthe growth and/or survival of a cell that is associated with aging,disease, or another condition (collectively “a target cell”), whichexpression cassettes, systems, and methods overcome the safety andefficacy concerns that are associated with existing technologies thatrely on targeted delivery of a therapeutic compound and, as a result of,for example, inefficient target cell delivery and/or off-target effects,have limited therapeutic benefit.

More specifically, the expression cassettes, systems, and methodsdisclosed herein exploit the cell-specific transcription regulatorymachinery that is intrinsic to a target cell and, thereby, achieve atarget cell-specific therapeutic benefit without the need fortargeted-delivery of a therapeutic compound. These expression cassettes,systems, and methods permit the target cell-specific induction ofexpression of a nucleic acid that encodes a therapeutic protein, whichprotein can reduce, prevent, and/or eliminate the growth and/or survivalof a cell in which it is produced.

Thus, the various embodiments that are provided by the presentdisclosure include:

-   -   1. Expression constructs for the targeted production of        therapeutic proteins within a target cell, such as a cell that        is associated with aging, disease, and/or another condition, the        expression construct comprising:        -   a. transcriptional promoter that is activated in response to            one or more factors each of which is produced within a            target cell and        -   b. a nucleic acid that is operably linked to and under            regulatory control of the transcriptional promoter, wherein            the nucleic acid encodes a therapeutic protein that can            reduce, prevent, and/or eliminate the growth and/or survival            of a cell, including the target cell.    -   2. Systems for the targeted production of a therapeutic protein        within a target cell, the systems comprising a vector for        delivering a nucleic acid to a cell, including a target cell as        well as a non-target cell,    -   wherein the vector comprises an expression construct for the        targeted production of a therapeutic protein within a target        cell (e.g., a cell that is associated with aging, cancer, and/or        other disease and/or condition) but not within a non-target        cell,    -   wherein the expression construct comprises (i) a transcriptional        promoter that is activated in response to one or more factors        each of which is produced within a target cell and (ii) a        nucleic acid that is operably linked to and under regulatory        control of the transcriptional promoter,    -   wherein the nucleic acid encodes a therapeutic protein that can        reduce, prevent, and/or eliminate the growth and/or survival of        a cell in which it is produced, including a target cell.    -   3. Methods for reducing, preventing, and/or eliminating the        growth of a target cell, the methods comprising contacting a        target cell with a system for the targeted production of a        therapeutic protein within a target cell,    -   wherein the system comprises a vector for delivery of a nucleic        acid to a cell,    -   wherein the vector comprises an expression construct for the        targeted production of a therapeutic protein within a target        cell (e.g., a cell that is associated with age, disease, or        other condition) but not within a non-target cell,    -   wherein the expression construct comprises (i) a transcriptional        promoter that is activated in response to one or more factors        each of which factors is produced within a target cell and (ii)        a nucleic acid that is operably linked to and under regulatory        control of the transcriptional promoter,    -   wherein the nucleic acid encodes a therapeutic protein that is        produced upon expression of the nucleic acid and    -   wherein production of the therapeutic protein in the target cell        (i.e., the cell that is associated with age, disease, or other        condition) reduces, prevents, and/or eliminates growth and/or        survival of the target cell.    -   4. Methods for the treatment of aging, disease, or other        condition in a human, wherein aging, disease, or other condition        is associated with a target cell, the methods comprising        administering to the human a system for the targeted production        of a therapeutic protein within a target cell,    -   wherein the system comprises a vector that is capable of        delivering a nucleic acid to a cell,    -   wherein the vector comprises an expression construct for the        targeted production of a therapeutic protein within a target        cell (e.g., a cell that is associated with age, disease, or        other condition) but not within a non-target cell,    -   wherein the expression construct comprises (i) a transcriptional        promoter that is activated in response to one or more factors        each of which factors is produced within a target cell and (ii)        a nucleic acid that is operably linked to and under regulatory        control of the transcriptional promoter,    -   wherein the nucleic acid encodes a therapeutic protein that is        produced upon expression of the nucleic acid and    -   wherein production of the therapeutic protein in the target cell        (i.e., the cell that is associated with age, disease, or other        condition) reduces, prevents, and/or eliminates growth and/or        survival of the target cell thereby slowing aging in the human        and/or slowing, reversing, and/or eliminating the disease or        condition in the human.

Definitions

These and other aspects of the present disclosure can be betterunderstood by reference to the following non-limiting definitions.

As used herein, the term “transcriptional promoter” refers to a regionof DNA that initiates transcription of a particular gene. Promoters arelocated near transcription start sites of genes, on the same strand andupstream on the DNA (towards the 3′ region of the anti-sense strand,also called template strand and non-coding strand). Promoters can beabout 100-1000 base pairs long. For the transcription to take place, theenzyme that synthesizes RNA, known as RNA polymerase, must attach to theDNA near a gene. Promoters contain specific DNA sequences and responseelements that provide a secure initial binding site for RNA polymeraseand for proteins called transcription factors that recruit RNApolymerase. These transcription factors have specific activator orrepressor sequences of corresponding nucleotides that attach to specificpromoters and regulate gene expressions. The process is morecomplicated, and at least seven different factors are necessary for thebinding of an RNA polymerase II to the promoter. Promoters representcritical elements that can work in concert with other regulatory regions(enhancers, silencers, boundary elements/insulators) to direct the levelof transcription of a given gene.

Eucaryotic transcriptional promoters comprise a number of essentialelements, which collectively constitute a core promoter (i.e., theminimal portion of a promoter that is required to initiatetranscription). Those elements include (1) a transcription start site(TSS), (2) an RNA polymerase binding site (in particular an RNApolymerase II binding site in a promoter for a gene encoding a messengerRNA), (3) a general transcription factor binding site (e.g., a TATA boxhaving a consensus sequence TATAAA, which is a binding site for aTATA-binding protein (TBP)), (4) a B recognition element (BRE), (5) aproximal promoter of approximately 250 bp that contains regulatoryelements, (6) transcription factor binding sites (e.g., an E-box havingthe sequence CACGTF, which is a binding site for basic helix-loop-helix(bHLH) transcription factors including BMAL11-Clock nad cMyc), and (7) adistal promoter containing additional regulatory elements. As usedherein, the term “transcriptional promoter” is distinct from the term“enhancer,” which refers to a regulatory element that is distant fromthe transcriptional start site.

Eucaryotic promoters are often categorized according to the followingclasses: (1) AT-based class, (2) CG-based class, (3) ATCG-compact class,(4) ATCG-balanced class, (5) ATCG-middle class, (6) ATCG-less class, (7)AT-less class, (8) CG-spike class, (9) CG-less class, and (10) ATspikeclass. See, Gagniuc and Ionescu-Tirgoviste, BMC Genomics 13:512 (2012).Eucaryotic promoters can be “unidirectional” or “bidirectional.”Unidirectional promoters regulate the transcription of a single gene andare characterized by the presence of a TATA box. Bidirectional promotersare short (<1 kbp), intergenic regions of DNA between the 5′ ends ofgenes in a bidirectional gene pair (i.e., two adjacent genes coded onopposite strands having 5′ ends oriented toward one another.Bidirectional genes are often functionally related and because theyshare a single promoter, can be co-regulated and co-expressed. Unlikeunidirectional promoters, bidirectional promoters do not contain a TATAbox but do contain GpC islands and exhibit symmetry around a midpoint ofdominant Cs and As on one side and Gs and Ts on the other. CCAAT boxesare common in bidirectional promoters as are NRF-1, GABPA, YY1, andACTACAnnTCCC motifs.

Transcriptional promoters often contain two or more transcription factorbinding sites. Thus, the efficient expression of a nucleic acid that isdownstream of a promoter having multiple transcription factor bindingsites typically requires the cooperative action of multipletranscription factors. Accordingly, the specificity of transcriptionalregulation, and hence expression of an associated nucleic acid, can beincreased by employing transcriptional promoters having two or moretranscription factor binding sites.

As used herein, the term “transcription factor” refers tosequence-specific DNA-binding factors that bind to specific sequenceswithin a transcriptional promoter thereby regulating the transcriptionof a nucleic acid that is in operable proximity to and downstream of thepromoter. Transcription factors include activators, which promotetranscription, and repressors, which block transcription by preventingthe recruitment or binding of an RNA polymerase. Transcription factorstypically contain (1) one or more DNA-binding domains (DBDs), whichfacilitate sequence specific binding to a cognate transcription factorbinding site (a/k/a response element) within a transcriptional promoter;(2) one or more signal-sensing domains (SSDs), which includes ligandbinding domains that are responsive to external signals; and (3) one ormore transactivation domains (TADs), which contain binding sites forother proteins, including transcription coregulators.

As used herein, the term “transcription factor” refers exclusively tothose factors having one or more DBDs and is not intended to includeother regulatory proteins such as coactivators, chromatin remodelers,histone acetylases, deacetylases, kinases, and methylases, which no notcontain DBDs.

Of the approximately 2,600 human proteins that contain DNA-bindingdomains, the majority are believed to be transcription factors.Transcription factors are categorized according to structural featuresof the DNA-binding domain, which include basic helix-loop-helix domains,basic-leucine zipper (bZIP domains), C-terminal effector domains ofbipartite response regulators, GCC box domains, helix-turn-helixdomains, homeodomains, lambda repressor-like domains, serum responsefactor-like (srf-like) domains, paired box domains, winged helixdomains, zinc finger domains, multi-Cys₂His₂ zinc finger domains,Zn₂Cys₆ domains, and Zn₂Cys₈ nuclear receptor zinc finger domains.

Many transcription factors are either tumor suppressors or oncogenes,and, thus, mutations within and the aberrant expression of suchtranscription factors is associated with some cancers and other diseasesand conditions. For example, transcription factors within (1) theNF-kappaB family, (2) the AP-1 family, (3) the STAT family, and (4) thesteroid receptor family have been implicated in the neurodevelopmentaldisorder Rett sysndrome (the MECP2 transcription factor), diabetes(hepatocyte nuclear factors (HNFs) and insulin promoter factor-1(IPF1/Pdx1)), developmental verbal dyspraxia (the FOXP2 transcriptionfactor), autoimmune diseases (the FOXP3 transcription factor),Li-Raumeni syndrome (the p53 tumor suppressor), and multiple cancers(the STAT and HOX family of transcription factors). Clevenger, Am. J.Pathol. 165(5):1449-60 (2004); Carrithers et al., Am J Pathol166(1):185-196 (2005); Herreros-Villanueve et al., World JGastroenterology 20(9):2247-2254 (2014); and Campbell et al., Am JPathol 158(1):25-32 (2001). Olsson et al., Oncogene 26(7):1028-37 (2007)describe the upregulation of the transcription factor E2F3, which is akey regulator of the cell cycle, in human bladder and prostate cancers.Cantile et al., Curr Med Chem 18(32):4872-84 (2011) describe theupregulation of HOX genes in urogenital cancers; Cillo et al., Int JCancer 129(11):2577-87 (2011) describe the upregulation of HOX genes inhepatocellular carcinoma; Cantile et al., Int J. Cancer 125(7):1532-41(2009) describe HOX D13 expression across 79 tumor tissue types; Cantileet al., J Cell Physiol 205(2):202-10 (2005) describe upregulation of HOXD expression in prostate cancers; Cantile et al., Oncogene 22(41):6462-8(2003) describe the hyperexpression of locus C genes in the HOX networkin human bladder transitional cell carcinomas; Morgan et al., BioMedCentral 14:15 (2014), describe HOX transcription factors as targets forprostate cancer; and Alharbi et al., Leukemia 27(5):1000-8 (2013)describe the role of HOXC genes in hematopoiesis and acute leukemia.

The AP-2 family includes five transcription factors that can act as bothrepressors and activators. AP-2γ regulates cancer cell survival byblocking p53 activation of the p21CIP gene. High levels of AP-2γ areassociated with poor prognosis in breast cancer. Gee et al., J Pathol217(1):32-41 (2009) and Williams et al., EMBO J 28(22):3591-601 (2009).A further transcription factor that promotes cell survival are theforkhead transcription factors (FOX), which can promote the expressionof proteins involved in drug resistance and also block programmed celldeath and may therefore protect cancer cells from chemotherapeuticdrugs. Gomes et al., Chin J. Cancer 32(7):365-70 (2013) describe therole of FOXO3a and FOXM1 in carcinogenesis and drug resistance.

Transcription factors can bind to promoters as well as to enhancers. Asused in the present disclosure, the term transcription factor refers tothe subset of transcription factors that bind to transcription factorbinding sites within a promoter and excludes those factors that bind toenhancer sequences. Transcription factors can also upregulate ordownregulate the expression of an associated nucleic acid. The presentdisclosure employs transcriptional promoters having transcription factorbinding sites for transcription factors that promote rather than inhibitexpression and therefore cause the upregulation in the expression of anassociated nucleic acid. Such transcription factors that upregulatenucleic acid expression include, for example and not limitation,transcription factors that (1) stabilize RNA polymerase binding to itscognate binding site, (2) recruit coactivator or corepressor proteins toa transcription factor DNA complex, and/or (3) catalyze the acetylationof histone proteins (or recruit one or more other proteins that catalyzethe acetylation of histone proteins). Such histone acetyltransferase(HAT) activity reduces the affinity of histone binding to DNA therebymaking the DNA more accessible for transcription.

As used herein, the term “necrosis” refers to a process leading to celldeath that occurs when a cell is damaged by an external force, such aspoison, a bodily injury, an infection, or loss of blood supply. Celldeath from necrosis causes inflammation that can result in furtherdistress or injury within the body. As used herein, the term “apoptosis”refers to a process leading to cell death in which a programmed sequenceof events leads to the elimination of cells without releasing harmfulsubstances. Apoptosis plays a crucial role in developing and maintainingthe health of the body by eliminating old cells, unnecessary cells, andunhealthy cells. Apoptosis is mediated by proteins produced by suicidegenes, including the caspase proteins, which break down cellularcomponents needed for survival and induce the production of DNAses,which destroy nuclear DNA.

As used herein, the term “suicide gene” refers to a class of genes thatproduce proteins that induce p53-mediated apoptotic cell killing.Suicide genes that can be employed in the expression constructs andsystems of the present disclosure include the caspases, Casp3, Casp8,Casp9, BAX, DFF40, Herpes Simplex Virus Thymidine Kinase (HSV-TK), andcytosine deaminase and inducible variants of Casp3, Casp8, Casp9, BAX,DFF40, Herpes HSV-TK, and cytosine deaminase.

The presently disclosed expression constructs and systems are used inmethods for the treatment of aging, cancer infectious disease, bacterialinfections, and/or other conditions as well as in methods for thekilling of cells that are associated with aging, cancer, infectiousdisease, bacterial infections, and/or other conditions and employ atherapeutic protein that reduces the growth and/or proliferation of atarget cell. In certain embodiments, the therapeutic protein can beexpressed by a suicide gene, which encodes Casp3, Casp8, Casp9, BAX,DFF40, HSV-TK, or cytosine deaminase as well as a inducible variants ofCasp3, Casp8, Casp9, BAX, DFF40, HSV-TK, or cytosine deaminase. Theexpression cassettes and systems can also be used in conjunction withconventional chemotherapeutics to enhance the effectiveness oftherapeutic regimen for the treatment of aging, cancers, infectiousdiseases, bacterial infections, and other diseases and conditions.

Within certain aspects of the present disclosure, expression constructsare pVAX1 (FIG. 14 ) plasmid expression constructs comprising apolynucleotide encoding a pro-apoptotic protein under the regulatorycontrol of a target cell-specific promoter, such as a senescentcell-specific promoter or a cancer cell-specific promoter.

Exemplary pVAX1™ plasmid expression constructs includepVAX-16s-iCasp9-MX (FIG. 16 ; SEQ ID NO: 6) for the target cell-specificexpression of an inducible Caspase 9 protein (iCasp9) under theregulatory control of a p16s promoter, pVAX1-53-iCasp9-MX (FIG. 26 ; SEQID NO: 7) for the target cell-specific expression of an inducibleCaspase 9 protein (iCasp9) under the regulatory control of a p53promoter, pVax1-p53-saCasp9-5 (FIG. 27 ; SEQ ID NO: 8) for the targetcell-specific expression of a self-activating Caspase 9 protein(saCASP9) under the regulatory control of a p53 promoter,pVax1-p53-iCasp9-OVA (FIG. 28 ; SEQ ID NO: 11) for the targetcell-specific expression of an inducible Caspase 9 protein (iCasp9) andan ovalbumin protein under the regulatory control of a p53 promoter,pVax1-p53-iCasp9-G-O (FIG. 29 ; SEQ ID NO: 9) for the targetcell-specific expression of an inducible Caspase 9 protein (iCasp9) andan ovalbumin protein under the regulatory control of a p53 promoter,pVax1-p53-iCasp9-huCD40L (FIG. 30 ; SEQ ID NO: 10) for the targetcell-specific expression of an inducible Caspase 9 protein (iCasp9) anda CD40 ligand protein (CD40L) under the regulatory control of a p53promoter.

Exemplary p10 plasmid expression constructs include p10-p16e-iCasp9(FIG. 17 ; SEQ ID NO: 12) for the target cell-specific expression of aninducible Caspase 9 protein (iCasp9) under the regulatory control of ap16e promoter, p10-p16e-saCasp9 (FIG. 18 ; SEQ ID NO: 13) for the targetcell-specific expression of a self-activating Caspase 9 protein(saCasp9) under the regulatory control of a p16e promoter,p10-p53-iCasp9 (FIG. 33 ; SEQ ID NO: 14) for the target cell-specificexpression of an inducible Caspase 9 protein (iCasp9) under theregulatory control of a p53 promoter, and p10-p53-saCasp9 (FIG. 34 ; SEQID NO: 15) for the target cell-specific expression of a self-activatingCaspase 9 protein (saCasp9) under the regulatory control of a p53promoter.

Within other aspects of the present disclosure, expression constructsare NTC-based plasmid expression constructs, including NTC8385, NTC8685,and NTC93 85 plasmid expression constructs, comprising a polynucleotideencoding a pro-apoptotic protein under the regulatory control of atarget cell-specific promoter, such as a senescent cell-specificpromoter or a cancer cell-specific promoter.

Within further aspects of the present disclosure, expression constructsare gWiz-based plasmid expression constructs comprising a polynucleotideencoding a pro-apoptotic protein under the regulatory control of atarget cell-specific promoter, such as a senescent cell-specificpromoter or a cancer cell-specific promoter.

The practice of the present disclosure will employ, unless indicatedspecifically to the contrary, conventional methodology and techniquesthat are in common use in the fields of virology, oncology, immunology,microbiology, molecular biology, and recombinant DNA, which methodologyand techniques are well known by and readily available to those havingskill of the art. Such methodology and techniques are explained fully inlaboratory manuals as well as the scientific and patent literature. See,e.g., Sambrook, et al., “Molecular Cloning: A Laboratory Manual” (2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989); Maniatis et al., “Molecular Cloning: A Laboratory Manual” (1982);“DNA Cloning: A Practical Approach, vol. I & II” (Glover, ed.);“Oligonucleotide Synthesis” (Gait, ed., 1984); Ausubel et al. (eds.),“Current Protocols in Molecular Biology” (John Wiley & Sons, 1994);“Nucleic Acid Hybridization” (Hames & Higgins, eds., 1985);“Transcription and Translation” (Hames & Higgins, eds., 1984); “AnimalCell Culture” (Freshney, ed., 1986); and Perbal, “A Practical Guide toMolecular Cloning” (1984). All publications, patents and patentapplications cited herein, whether supra or infra, are herebyincorporated by reference in their entirety.

Systems and Expression Constructs for Reducing, Preventing, and/orEliminating the Growth and/or Survival of a Target Cell

Within certain embodiments, the present disclosure provides expressionconstructs and systems comprising a delivery vector and an expressionconstruct for achieving a target cell specific reduction, prevention,and/or elimination in the growth and/or survival of the target cell.

Systems

Systems of the present disclosure comprise (1) a vector that is capableof non-specific delivery of a nucleic acid to a cell, whether that cellis a target cell or a non-target cell, and (b) an expression constructcomprising a target cell specific transcriptional promoter and a nucleicacid that encodes a therapeutic protein, which expression constructsachieve the target cell specific production of a therapeutic protein.The systems disclosed herein will find utility in a broad range oftherapeutic applications in which it is desirable to effectuate thegrowth or survival characteristics of a target cell, such as a cell thatis associated with aging, disease, or another condition, but, at thesame time, to not effectuate the growth or survival characteristics of anormal, a non-target cell that is not associated with aging, disease, oranother condition.

The present disclosure provides systems for effectuating the growthand/or survival of a broad range of cells that are associated withaging, disease, or other conditions that similarly comprises (1) anon-specific nucleic acid delivery vector and (2) an expressionconstruct comprising (a) a target cell specific transcriptional promoterand (b) a nucleic acid that encodes a therapeutic protein. Each of theseaspects of the presently disclosed systems are described in furtherdetail herein.

Within certain embodiments, provided herein are systems for effectuatingthe growth and/or survival of target cells, which systems comprise: (1)a non-specific nucleic acid delivery vector and (2) an expressionconstruct comprising: (a) a transcriptional promoter, whichtranscriptional promoter is activated in target cells but not in normal,non-target cells, and (b) a nucleic acid that is under the control ofthe transcriptional promoter, which nucleic acid encodes a therapeuticprotein that can reduce, prevent, and/or eliminate the growth and/orsurvival of a target cell, for example by inducing a mechanism ofprogrammed cell death in a cell in which it is produced. Thus, thesesystems achieve the selective killing of target cells by exploitingtranscriptional machinery that is produced in, and intrinsic to, targetcells; without the use of toxins and in the absence of target cellspecific delivery of the expression construct.

In certain aspects of these embodiments wherein the human target cell isa senescent cell, the transcriptional promoter can include at least atranscription factor binding site (i.e., a response element) ofp16INK4a/CDKN2A as described in Wang et al., J. Biol. Chem.276(52):48655-61 (2001), which transcriptional promoter is responsive toactivation by a factor such as SP1, ETS1, and ETS2. The transcriptionalpromoter can also include at least a transcription factor binding site(i.e., a response element) of p21/CDKN1A, which transcriptional promoteris responsive to activation by a factor such as p53/TP53.Transcriptional activation induces the expression of a nucleic acid thatencodes a therapeutic protein such as Casp3, Casp8, Casp9, DFF40, BAX,HSV-TK, or carbonic anhydrase or an inducible variant of Casp3, Casp8,Casp9, BAX, DFF40, HSV-TK, or cytosine deaminase.

In other aspects of these embodiments wherein the human target cell is acancer cell, such as a brain cancer cell, a prostate cancer cell, a lungcancer cell, a colorectal cancer cell, a breast cancer cell, a livercancer cell, a hematologic cancer cell, and a bone cancer cell, thetranscriptional promoter can include at least a transcription factorbinding site (i.e., a response element) of the p21^(cip1/waf1) promoter,the p27^(kip1) promoter, the p57^(kip2) promoter, the TdT promoter, theRag-1 promoter, the B29 promoter, the Blk promoter, the CD19 promoter,the BLNK promoter, and/or the λ5 promoter, which transcriptionalpromoter is responsive to activation by one or more transcriptionfactors such as an EBF3, O/E-1, Pax-5, E2A, p53, VP16, MLL, HSF1,NF-IL6, NFAT1, AP-1, AP-2, HOX, E2F3, and/or NF-κB transcription factor,and which transcriptional activation induces the expression of a nucleicacid that encodes a therapeutic protein such as, for example, Casp3,Casp8, Casp9, BAX, DFF40, HSV-TK, or cytosine deaminase or an induciblevariant of Casp3, Casp8, Casp9, BAX, DFF40, HSV-TK, or cytosinedeaminase which therapeutic protein reduces, prevents, and/or eliminatesthe growth and/or survival of the cancer cell, such as, for example, byinducing cell death in the senescent cell via a cellular processincluding apoptosis. Other therapeutic proteins may be employed thatreduce, prevent, and/or eliminate the growth and/or survival of a cancercell by, for example, inducing cell death via a cellular processincluding necrosis/necroptosis, autophagic cell death, endoplasmicreticulum-stress associated cytotoxicity, mitotic catastrophe,paraptosis, pyroptosis, pyronecrosis, and entosifs. In still furtheraspects of these embodiments wherein the target cell is a human cellthat is infected with an infectious agent, such as a virus, including,for example, a herpes virus, a polio virus, a hepatitis virus, aretrovirus, an influenza virus, and a rhino virus, or the target cell isa bacterial cell, the transcriptional promoter can be activated by afactor that is expressed by the infectious agent or bacterial cell,which transcriptional activation induces the expression of a nucleicacid that encodes a therapeutic protein such as, for example, Casp3,Casp8, Casp9, BAX, DFF40, HSV-TK, or cytosine deaminase or an induciblevariant of Casp3, Casp8, Casp9, BAX, DFF40, HSV-TK, or cytosinedeaminase which therapeutic protein reduces, prevents, and/or eliminatesthe growth and/or survival of the senescent cell, such as, for example,by inducing cell death in the senescent cell via a cellular processincluding apoptosis. Other therapeutic proteins may be employed thatreduce, prevent, and/or eliminate the growth and/or survival of asenescent cell by, for example, inducing cell death via a cellularprocess including necrosis/necroptosis, autophagic cell death,endoplasmic reticulum-stress associated cytotoxicity, mitoticcatastrophe, paraptosis, pyroptosis, pyronecrosis, and entosifs.

Each of these aspects of the presently disclosed systems are describedin further detail herein.

1. Non-specific Nucleic Acid Delivery Vectors

The systems of the present disclosure achieve target cell specificity byexploiting transcriptional machinery that is unique to a target cell.Thus, the systems described herein employ nucleic acid delivery vectorsthat can be readily adapted for the non-specific delivery of expressionconstructs to a cell, including but not limited to a target cell.

A wide variety of both non-viral and viral nucleic acid delivery vectorsare well known and readily available in the art and may be adapted foruse for the non-specific cellular delivery of the expression constructsdisclosed herein. See, for example, Elsabahy et al., Current DrugDelivery 8(3):235-244 (2011) for a general description of viral andnon-viral nucleic acid delivery methodologies. The successful deliveryof a nucleic acid into mammalian cells relies on the use of efficientdelivery vectors. Viral vectors exhibit desirable levels of deliveryefficiency, but often also exhibit undesirable immunogenicity,inflammatory reactions, and problems associated with scale-up, all ofwhich can limit their clinical use. The ideal vectors for the deliveryof a nucleic acid are safe, yet ensure nucleic acid stability and theefficient transfer of the nucleic acid to the appropriate cellularcompartments.

Non-limiting examples of non-viral and viral nuclic acid deliveryvectors are described herein and disclosed in scientific and patentliterature. More specifically, the presently disclosed systems mayemploy one or more liposomal vectors, viral vectors, nanoparticles,polyplexesm dendrimers, each of which has been developed for thenon-specific delivery of nucleic acids, can be adapted for thenon-specific delivery of the expression constructs described herein, andcan be modified to incorporate one or more agents for promoting thetargeted delivery of a system to a target cell of interest therebyenhancing the target cell specificity of the presently disclosedsystems.

2. Liposomal Vectors and Nanoparticles

An expression cassette may be incorporated within and/or associated witha lipid membrane, a lipid bi-layer, and/or a lipid complex such as, forexample, a liposome, a vesicle, a micelle and/or a microsphere. Suitablemethodology for preparing lipid-based delivery systems that may beemployed with the expression constructs of the present disclosure aredescribed in Metselaar et al., Mini Rev. Med. Chem. 2(4):319-29 (2002);O'Hagen et al., Expert Rev. Vaccines 2(2):269-83 (2003); O'Hagan, Curr.Durg Targets Infect. Disord. 1(3):273-86 (2001); Zho et al., Biosci Rep.22(2):355-69 (2002); Chikh et al., Biosci Rep. 22(2):339-53 (2002);Bungener et al., Biosci. Rep. 22(2):323-38 (2002); Park, Biosci Rep.22(2):267-81 (2002); Ulrich, Biosci. Rep. 22(2):129-50; Lofthouse, Adv.Drug Deliv. Rev. 54(6):863-70 (2002); Zhou et al., J. Immunother.25(4):289-303 (2002); Singh et al., Pharm Res. 19(6):715-28 (2002); Wonget al., Curr. Med. Chem. 8(9):1123-36 (2001); and Zhou et al.,Immunomethods 4(3):229-35 (1994). Midoux et al., British J. Pharmacol157:166-178 (2009) describe chemical vectors for the delivery of nucleicacids including polymers, peptides and lipids. Sioud and Sorensen,Biochem Biophys Res Commun 312(4):1220-5 (2003) describe cationicliposomes for the delivery of nucleic acids.

Due to their positive charge, cationic lipids have been employed forcondensing negatively charged DNA molecules and to facilitate theencapsulation of DNA into liposomes. Cationic lipids also provide a highdegree of stability to liposomes. Cationic liposomes interact with acell membrane and are taken up by a cell through the process ofendocytosis. Endosomes formed as the results of endocytosis, are brokendown in the cytoplasm thereby releasing the cargo nucleic acid. Becauseof the inherent stability of cationic liposomes, however, transfectionefficiencies can be low as a result of lysosomal degradation of thecargo nucleic acid.

Helper lipids (such as the electroneutral lipid DOPE and L-a-dioleoylphosphatidyl choline (DOPC)) can be employed in combination withcationic lipids to form liposomes having decreased stability and,therefore, that exhibit improved transfection efficiencies. Theseelectroneutral lipids are referred to as Fusogenix lipids. See, Gruneret al., Biochemistry 27(8):2853-66 (1988) and Farhood et al., BiochimBiophys Acta 1235(2):289-95 (1995). DOPE forms an HII phase structurethat induces supramolecular arrangements leading to the fusion of alipid bilayer at a temperature greater than 5° C. to 10° C. Theincorporation of DOPE into liposomes also helps the formation of HIIphases that destabilize endosomal membranes.

Cholesterol can be employed in combination with DOPE liposomes forapplications in which a liposomal vector is administered intravenously.Sakurai et al., Eur J Pharm Biopharm 52(2):165-72 (2001). The presenceof one unsaturation in the acyl chain of DOPE is a crucial factor formembrane fusion activity. Talbot et al., Biochemistry 36(19):5827-36(1997).

Fluorinated helper lipids having saturated chains, such as DF4C11PE(rac-2,3-Di[11-(F-butyl)undecanoyl) glycero-1-phosphoethanolamine) alsoenhance the transfection efficiency of lipopolyamine liposomes. Boussifet al., J Gene Med 3(2):109-14 (2001); Gaucheron et al., Bioconj Chem12(6):949-63 (2001); and Gaucheron et al., J Gene Med 3(4):338-44(2001).

The helper lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)enhances efficient of in vitro cell transfection as compared to DOPElipoplexes. Prata et al., Chem Commun 13:1566-8 (2008). Replacement ofthe double bond of the oleic chains of DOPE with a triple bond as inDistear-4-ynoyl L-a-phosphatidylethanolamine [DS(9-yne)PE] has also beenshown to produce more stable lipoplexes. Fletcher et al., Org BiomolChem 4(2):196-9 (2006).

Amphiphilic anionic peptides that are derived from the N-terminalsegment of the HA-2 subunit of influenza virus haemagglutinin, such asthe IFN7 (GLFEAIEGFIE NGWEGMIDGW YG) and ESCA (GLFEAIAEFI EGGWEGLIEG CA)peptides, can be used to increase the transfection efficiency ofliposomes by several orders of magnitude. Wagner et al., Proc Natl AcadSci U.S.A. 89(17):7934-8 (1992); Midoux et al., Nucl Acids Res.21(4):871-8 (1993); Kichler et al., Bioconjug Chem 8(2):213-21 (1997);Wagner, Adv Drug Deliv Rev 38(3):279-289 (1999); Zhang et al., J GeneMed 3(6):560-8 (2001). Some artificial peptides such as GALA have beenalso used as fusogenic peptides. See, for example, Li et al., Adv DrugDeliv Rev 56(7):967-85 (2004) and Sasaki et al., Anal Bioanal Chem391(8):2717-27 (2008). The fusogenic peptide of the glycoprotein H fromherpes simplex virus improves the endosomal release of DNA/Lipofectaminelipoplexes and transgene expression in human cell (Tu and Kim, J GeneMed 10(6):646-54 (2008).

PCT Patent Publication Nos. WO 1999024582A1 and WO 2002/044206 describea class of proteins derived from the family Reoviridae that promotemembrane fusion. These proteins are exemplified by the p14 protein fromreptilian reovirus and the p16 protein from aquareovirus. PCT PatentPublication No. WO 2012/040825 describes recombinant polypeptides forfacilitating membrane fusion, which polypepides have at least 80%sequence identity with the ectodomain of p14 fusion-associated smalltransmembrane (FAST) protein and having a functional myristoylationmotif, a transmembrane domain from a FAST protein and a sequence with atleast 80% sequence identity with the endodomain of p15 FAST protein. The'825 PCT further describes the addition of a targeting ligand to therecombinant polypeptide for selective fusion. The recombinantpolypeptides presented in the '825 PCT can be incorporated within themembrane of a liposome to facilitate the delivery of nucleic acids.Fusogenix liposomes for delivering therapeutic compounds, includingnucleic acids, to the cytoplasm of a mammalian cell, which reduceliposome disruption and consequent systemic dispersion of the cargonucleic acid and/or uptake into endosomes and resulting nucleic aciddestruction are available commercially from Innovascreen Inc. (Halifax,Nova Scotia, CA).

A wide variety of inorganic nanoparticles, including gold, silica, ironoxide, titanium, hydrogels, and calcium phosphates have been describedfor the delivery of nucleic acids and can be adapted for the delivery ofthe expression constructs described herein. See, for example Wagner andBhaduri, Tissue Engineering 18(1):1-14 (2012) (describing inorganicnanoparticles for delivery of nucleic acid sequences); Ding et al., MolTher e-pub (2014) (describing gold nanoparticles for nucleic aciddelivery); Zhang et al., Langmuir 30(3):839-45 (2014) (describingtitanium dioxide nanoparticles for delivery of DNA oligonucleotides);Xie et al., Curr Pharm Biotechnol 14(10):918-25 (2014) (describingbiodegradable calcium phosphate nanoparticles fro gene delivery); Sizovset al., J Am Chem Soc 136(1):234-40 (2014) (describing sub-30monodisperse oligonucleotide nanoparticles).

Among the advantages of inorganic vectors are their storage stability,low immunogenicity, and resistance to microbial attack. Nanoparticles ofless than 100 nm can efficiently trap nucleic acids and allows itsescape from endosomes without degradation. Inorganic nanoparticlesexhibit improved in vitro transfection for attached cell lines due totheir high density and preferential location on the base of the culturedish. Quantum dots have been described that permit the coupling ofnucleic acid delivery with stable fluorescence markers.

Hydrogel nanoparticles of defined dimensions and compositions, can beprepared via a particle molding process referred to as PRINT (ParticleReplication in Non-wetting Templates), and can be used as deliveryvectors for the expression constructs disclosed herein. Nucleic acidscan be encapsulated in particles through electrostatic association andphysical entrapment. To prevent the disassociation of cargo nucleicacids from nanoparticles following systemic administration, apolymerizable conjugate with a degradable, disulfide linkage can beemployed.

The PRINT technique permits the generation of engineered nanoparticleshaving precisely controlled properties including size, shape, modulus,chemical composition and surface functionality for enhancing thetargeting of the expression cassette to a target cell. See, e.g., Wanget al., J Am Chem Soc 132:11306-11313 (2010); Enlow et al., Nano Lett11:808-813 (2011); Gratton et al., Proc Natl Acad Sci USA105:11613-11618 (2008); Kelly, J Am Chem Soc 130:5438-5439 (2008);Merkel et al. Proc Natl Acad Sci USA 108:586-591 (2011). PRINT is alsoamenable to continuous roll-to-roll fabrication techniques that permitthe scale-up of particle fabrication under good manufacturing practice(GMP) conditions.

Nanoparticles can be encapsulated with a lipid coating to improve oralbioavailability, minimize enzymatic degradation and cross blood brainbarrier. The nanoparticle surface can also be PEGylated to improve watersolubility, circulation in vivo, and stealth properties.

3. Viral Vectors

A wide variety of viral vectors are well known by and readily availableto those of skill in the art, including, for example, herpes simplexviral vectors lentiviral vectors, adenoviral vectors, andadeno-associated viral vectors, which viral vectors can be adapted foruse in the systems disclosed herein for the delivery of nucleic acids,in particular nucleic acids comprising an expression cassette for thetarget cell specific expression of a therapeutic protein.

The tropisms of natural or engineered viruses towards specific receptorsare the foundations for constructing viral vectors for delivery ofnucleic acids. The attachment of these vectors to a target cell iscontingent upon the recognition of specific receptors on a cell surfaceby a ligand on the viral vector. Viruses presenting very specificligands on their surfaces anchor onto the specific receptors on a cell.Viruses can be engineered to display ligands for receptors presented onthe surface of a target cell of interest. The interactions between cellreceptors and viral ligands are modulated in vivo by toll likereceptors.

The entry of a viral vector into a cell, whether via receptor mediatedendocytosis or membrane fusion, requires a specific set of domains thatpermit the escape of the viral vector from endosomal and/or lysosomalpathways. Other domains facilitate entry into nuclei. Replication,assembly, and latency determine the dynamics of interactions between thevector and the cell and are important considerations in the choice of aviral vector, as well as in engineering therapeutic cargo carryingcells, in designing cancer suicide gene therapies.

Herpes simplex virus (HSV) belongs to a family of herpesviridae, whichare enveloped DNA viruses. HSV binds to cell receptors through orthologsof their three main ligand glycoproteins: gB, gH, and gL, and sometimesemploy accessory proteins. These ligands play decisive roles in theprimary routes of virus entry into oral, ocular, and genital forms ofthe disease. HSV possesses high tropism towards cell receptors of thenervous system, which can be utilized for engineering recombinantviruses for the delivery of expression cassettes to target cells,including senescent cells, cancer cells, and cells infected with aninfectious agent. Therapeutic bystander effects are enhanced byinclusion of connexin coding sequences into the constructs. HerpesSimplex Virus vectors for the delivery of nucleic acids to target cellshave been reviewed in Anesti and Coffin, Expert Opin Biol Ther10(1):89-103 (2010); Marconi et al., Adv Exp Med Biol 655:118-44 (2009);and Kasai and Saeki, Curr Gene Ther 6(3):303-14 (2006).

Lentivirus belongs to a family of retroviridae, which are enveloped,single stranded RNA retroviruses and include the Human immunodeficiencyvirus (HIV). HIV envelope protein binds CD4, which is present on thecells of the human immune system such as CD4+ T cells, macrophages, anddendritic cells. Upon entry into a cell, the viral RNA genome is reversetranscribed into double-stranded DNA, which is imported into the cellnucleus and integrated into the cellular DNA. HIV vectors have been usedto deliver the therapeutic genes to leukemia cells. Recombinantlentiviruses have been described for mucin-mediated delivery of nucleicacids into pancreatic cancer cells, to epithelial ovarian carcinomacells, and to glioma cells, without substantial non-specific delivery tonormal cells. Lentiviral vectors for the delivery of nucleic acids totarget cells have been reviewed in Primo et al., Exp Dermatol21(3):162-70 (2012); Staunstrup and Mikkelsen, Curr Gene Ther11(5):350-62 (2011); and Dreyer, Mol Biotechnol 47(2):169-87 (2011).

Adenovirus is a non-enveloped virus consisting of a double-stranded,linear DNA genome and a capsid. Naturally, adenovirus resides inadenoids and may be a cause of upper respiratory tract infections.Adenovirus utilizes a cell's coxsackie virus and adenovirus receptor(CAR) for the adenoviral fiber protein for entry into nasal, tracheal,and pulmonary epithelia. CARs are expressed at low levels on senescentand cancer cells. Recombinant adenovirus can be generated that arecapable of nucleic acid deliver to target cells. Replication-competentadenovirus-mediated suicide gene therapy (ReCAP) is in the clinicaltrials for newly-diagnosed prostate cancer. Adenoviral vectors for thedelivery of nucleic acids to target cells have been reviewed in Huangand Kamihira, Biotechnol Adv. 31(2):208-23 (2013); Alemany, Adv CancerRes 115:93-114 (2012); Kaufmann and Nettelbeck, Trends Mol Med18(7):365-76 (2012); and Mowa et al., Expert Opin Drug Deliv7(12):1373-85 (2010).

Adeno-associated virus (AAV) is a small virus that infects humans andsome other primate species. AAV is not currently known to cause diseaseand consequently the virus causes a very mild immune response. Vectorsusing AAV can infect both dividing and quiescent cells and persist in anextrachromosomal state without integrating into the genome of the hostcell. These features make AAV a very attractive candidate for creatingviral vectors for use in the systems of the present disclosure.Adeno-associated virus (AAV) vectors for the delivery of nucleic acidsto target cells have been reviewed in Li et al., J. Control Release172(2):589-600 (2013); Hajitou, Adv Genet 69:65-82 (2010); McCarty, MolTher 16(10):1648-56 (2008); and Grimm et al., Methods Enzymol392:381-405 (2005).

4. Polyplexes

Polyplexes are complexes of polymers with DNA. Polyplexes consist ofcationic polymers and their fabrication is based on self-assembly byionic interactions. One important difference between the methods ofaction of polyplexes and liposomes and lipoplexes is that polyplexescannot directly release their nucleic acid cargo into the cytoplasm of atarget cell. As a result co-transfection with endosome-lytic agents suchas inactivated adenovirus is required to facilitate escape from theendocytic vesicle made during particle uptake. better understanding ofthe mechanisms by which DNA can escape from endolysosomal pathway (i.e.,the proton sponge effect) has triggered new polymer synthesis strategiessuch as the incorporation of protonable residues in polymer backbone andhas revitalized research on polycation-based systems. See, e.g.,Parhamifar et al., Methods e-pub (2014); Rychgak and Kilbanov, Adv DrugDeliv Rev e-pub (2014); Jafari et al., Curr Med Chem 19(2):197-208(2012).

Due to their low toxicity, high loading capacity, and ease offabrication, polycationic nanocarriers exhibit substantial advantagesover viral vectors, which show high immunogenicity and potentialcarcinogenicity and lipid-based vectors which cause dose dependenttoxicity. Polyethyleneimine, chitosan, poly(beta-amino esters), andpolyphosphoramidate have been described for the delivery of nucleicacids. See, e.g., Buschmann et al., Adv Drug Deliv Rev 65(9):1234-70(2013). The size, shape, and surface chemistry of these polymericnano-carriers can be easily manipulated.

5. Dendrimers

Dendrimers are highly branched macromolecules having a spherical shape.The surface of dendrimer particles may be functionalized such as, forexample, with positive surface charges (cationic dendrimers), which maybe employed for the delivery of nucleic acids. Dendrimer-nucleic acidcomplexes are taken into a cell via endocytosis. Dendrimers offer robustcovalent construction and extreme control over molecule structure andsize. Dendrimers are available commercially from DendriticNanotechnologies Inc. (Priostar; Mt Pleasant, MI), who producedendrimers using kinetically driven chemistry, which can be adapted frothe delivery of nucleic acids and can transfect cells at a highefficiency with low toxicity.

It will be understood that, while targeted delivery of an expressionconstruct is not required by the systems of the present disclosure andthat the targeted reduction, prevention, and/or elimination in thegrowth and/or survival of a target cell may be achieved by exploitingthe intracellular transcriptional machinery of a target cell that isunique to the target cell, it may be desireable, depending upon theprecise application contemplated, the incorporate into an otherwisenon-specific delivery vector one or more components that facilitate thetargeted delivery to a subset of cells at least some of which include atarget cell that is susceptible to the growth and/or survival inhibitionby the expression constructs of the present disclosure.

The targeted delivery of nucleic acids by liposome, nanoparticle, viraland other vectors described herein has been described in the scientificand patent literature and is well known by and readily available tothose of skill in the art. Such targeted delivery technologies may,therefore, be suitably adapted for targeting the delivery of expressionconstructs of the present disclosure to enhance the specificity of thegrowth and/or survival reduction, prevention, and/or elimination that isachieved within a target cell. The following examples of targeteddelivery technologies are provided herein to exemplify, not to limit,the targeted delivery vectors that may be adapted to achieve the systemsof the present disclosure.

Expression Constructs

Expression constructs of the present disclosure comprise: (a) atranscriptional promoter that is responsive to a factor or factors thatare produced in a target cell, one or more of which factors is notproduced, is produced at a substantially reduced level, is inactive,and/or exhibits a substantially reduced activity in a non-target cell;and (b) a nucleic acid that is operably linked to and under theregulatory control of the transcriptional promoter, wherein the nucleicacid encodes a protein that is capable of reducing, preventing, and/oreliminating the growth and/or survival of a cell in which it isproduced, including a target cell.

1. Target Cell Specific Transcriptional Promoters

The present disclosure provides systems comprising a vector fordelivering a nucleic acid to a cell wherein the nucleic acid is underthe transcriptional control of a promoter that is derepressed oractivated in a target cell, but is reprepressed or inactivated in anormal cell, non-target cell.

It will be understood the specificity of the presently disclosed systemstoward a target cell is achieved, therefore, through the targetcell-specific transcriptional activation of a nucleic acid that encodesa protein that reduces, prevents, and/or eliminates the growth and/orsurvival of a cell without regard to whether that cell is a target cell.Thus, the target cell specificity of the presently-disclosed systemsderives from the transcriptional promoter that regulates the expressionof the nucleic acid within the expression cassette in conjunction withtranscription-regulatory machinery that is provided by, and unique to,the target cell.

Thus, transcriptional promoters that may be suitably employed in theexpression constructs, systems, and methods of the present disclosureinclude those transcriptional promoters that are capable of promotingthe expression of a nucleic acid in a target cell (i.e., a cell that isassociated with aging, disease, or other condition), but incapable of,or exhibit a substantially reduced capability of, promoting expressionof that nucleic acid in a non-target cell.

Exemplified herein are expression constructs and systems comprisingexpression constructs wherein the transcriptional promoter is activatedin a target cell that is associated with aging, disease, or anothercondition.

In some embodiments, the present disclosure provides expressionconstructs and systems that may be employed in methods for the treatmentof aging reducing, preventing, and/or eliminating the growth and/orsurvival of a cell, such as a senescent cell, which is associated withaging. In certain aspects of those embodiments, expression constructsemploy a transcriptional promter that is responsive to one or morefactors that are produced within a target cell, such as a senescentcell, but are not produced in a non-target cell wherein those one ormore factors derepress and/or activate the transcriptional promoter and,as a consequence, promote the expression of a nucleic acid encoding atherapeutic protein that reduces, prevents, and/or eliminates the growthand/or survival of a cell that is associated with aging, including asenescent cell.

The transcriptional promoter itself is the primary mechanism by whichsenescent cells are preferentially targeted by the systems described inthis disclosure. A prototypic example of a target specifictranscriptional promoter for use with the systems in this disclosure isa promoter that is only active or mostly active in senescent cells. Anumber of promoters known by artisans to be active in senescent cellsmay be used with this system.

In certain aspects of these embodiments wherein the human target cell isa senescent cell, the transcriptional promoter can include the promoterregion of p16INK4a/CDKN2A as described in Wang et al., J. Biol. Chem.276(52):48655-61 (2001), which transcriptional promoter is responsive toactivation by a factor such as SP1, ETS1, and ETS2. The transcriptionalpromoter can also include the promoter region of p21/CDKN1A, whichtranscriptional promoter is responsive to activation by a factor such asp53/TP53.

In other aspects of these embodiments wherein the human target cell is acancer cell, such as a brain cancer cell, a prostate cancer cell, a lungcancer cell, a colorectal cancer cell, a breast cancer cell, a livercancer cell, a hematologic cancer cell, and a bone cancer cell, thetranscriptional promoter can include the p21^(cip1/waf1) promoter, thep27^(kip1) promoter, the p57^(kip2) promoter, the TdT promoter, theRag-1 promoter, the B29 promoter, the Blk promoter, the CD19 promoter,the BLNK promoter, and/or the λ5 promoter, which transcriptionalpromoter is responsive to activation by one or more transcriptionfactors such as an EBF3, O/E-1, Pax-5, E2A, p53, VP16, MLL, HSF1,NF-IL6, NFAT1, AP-1, AP-2, HOX, E2F3, and/or NF-κB transcription factor,and which transcriptional activation induces the expression of a nucleicacid that encodes a therapeutic protein.

In still further aspects of these embodiments wherein the target cell isa human cell that is infected with an infectious agent, such as a virus,including, for example, a herpes virus, a polio virus, a hepatitisvirus, a retrovirus virus, an influenza virus, and a rhino virus, or thetarget cell is a bacterial cell, the transcriptional promoter can beactivated by a factor that is expressed by the infectious agent orbacterial cell, which transcriptional activation induces the expressionof a nucleic acid that encodes a therapeutic protein.

2. The p16 Transcriptional Promoter

In one embodiment, the suicide gene could be placed under control of ap16 promoter, such as a p16Ink4a gene promoter, which istranscriptionally active in senescent, but not in non-senescent cells.

In humans, p16 is encoded by the CDKN2A gene, which gene is frequentlymutated or deleted in a wide variety of tumors. p16 is an inhibitor ofcyclin dependent kinases such as CDK4 and CDK6, which phosphorylateretinoblastoma protein (pRB) thereby causing the progression from G1phase to S phase. p16 plays an important role in cell cycle regulationby decelerating cell progression from G1 phase to S phase, and thereforeacts as a tumor suppressor that is implicated in the prevention ofcancers, including, for example, melanomas, oropharyngeal squamous cellcarcinomas, and esophageal cancers. The designation p16Ink4A refers tothe molecular weight (15,845) of the protein encoded by one of thesplice variants of the CDKN2A gene and to its role in inhibiting CDK4.

In humans, p16 is encoded by CDKN2A gene, located on chromosome 9(9p21.3). This gene generates several transcript variants that differ intheir first exons. At least three alternatively spliced variantsencoding distinct proteins have been reported, two of which encodestructurally related isoforms known to function as inhibitors of CDK4.The remaining transcript includes an alternate exon 1 located 20 kbupstream of the remainder of the gene; this transcript contains analternate open reading frame (ARF) that specifies a protein that isstructurally unrelated to the products of the other variants. The ARFproduct functions as a stabilizer of the tumor suppressor protein p53,as it can interact with and sequester MDM2, a protein responsible forthe degradation of p53. In spite of their structural and functionaldifferences, the CDK inhibitor isoforms and the ARF product encoded bythis gene, through the regulatory roles of CDK4 and p53 in cell cycle G1progression, share a common functionality in control of the G1 phase ofthe cell cycle. This gene is frequently mutated or deleted in a widevariety of tumors and is known to be an important tumor suppressor gene.

Concentrations of p16INK4a increase dramatically as tissue ages. Liu etal., Aging Cell 8(4):439-48 (2009) and Krishnamurthy et al., Nature443(7110):453-7 (2006). The increased expression of the p16 gene withage reduces the proliferation of stem cells thereby increasing thecellular senescence-associated health risks in a human.

p16 is a cyclin-dependent kinase (CDK) inhibitor that slows down thecell cycle by prohibiting progression from G1 phase to S phase.Normally, CDK4/6 binds cyclin D and forms an active protein complex thatphosphorylates retinoblastoma protein (pRB). Once phosphorylated, pRBdisassociates from the transcription factor E2F1, liberating E2F1 fromits cytoplasm bound state allowing it to enter the nucleus. Once in thenucleus, E2F1 promotes the transcription of target genes that areessential for transition from G1 to S phase.

p16 acts as a tumor suppressor by binding to CDK4/6 and preventing itsinteraction with cyclin D. This interaction ultimately inhibits thedownstream activities of transcription factors, such as E2F1, andarrests cell proliferation. This pathway connects the processes of tumoroncogenesis and senescence, fixing them on opposite ends of a spectrum.

On one end, the hypermethylation, mutation, or deletion of p16 leads todownregulation of the gene and can lead to cancer through thedysregulation of cell cycle progression. Conversely, activation of p16through the ROS pathway, DNA damage, or senescence leads to the build upof p16 in tissues and is implicated in aging of cells.

Regulation of p16 is complex and involves the interaction of severaltranscription factors, as well as several proteins involved inepigenetic modification through methylation and repression of thepromoter region. PRC1 and PRC2 are two protein complexes that modify theexpression of p16 through the interaction of various transcriptionfactors that execute methylation patterns that can repress transcriptionof p16. These pathways are activated in cellular response to reducesenescence.

3. The p21 Transcriptional Promoter

A nucleic acid encoding a therapeutic protein could be placed under thecontrol of the p21/CDKN1A transcriptional promoter that is oftentranscriptionally active in senescent, and cancerous or pre-cancerouscells. p53/TP53 plays a central role in the regulation of p21 and,therefore, in the growth arrest of cells when damaged. p21 protein bindsdirectly to cyclin-CDK complexes that drive the cell cycle and inhibitstheir kinase activity thereby causing cell cycle arrest to allow repairto take place. p21 also mediates growth arrest associated withdifferentiation and a more permanent growth arrest associated withcellular senescence. The p21 gene contains several p53 response elementsthat mediate direct binding of the p53 protein, resulting intranscriptional activation of the gene encoding the p21 protein. Therole of p53 gene regulation in cellular senescence is described inKelley et al., Cancer Research 70(9):3566-75. (2010).

Nucleic Acids and Therapeutic Proteins Encoded Thereby

Nucleic acids that may be suitably employed in the expressionconstructs, systems, and methods of the present disclosure encode aprotein that is capable of reducing, preventing, and/or eliminating thegrowth and/or survival of a cell in which it is produced, including atarget cell. Thus, the target cell specificity of the presentlydisclosed expression constructs and systems is achieved by theexpression within a target cell, but not within a non-target cell, of anucleic acid that encodes a therapeutic protein.

Nucleic acids encoding therapeutic proteins that may be employed in theexpression constructs and systems of the present disclosure includenucleic acids encoding one or more protein that induces apoptosis in acell in which it is produced. Exemplified herein are expressionconstructs and systems comprising one or more “suicide genes,” such as anucleic acid encoding Herpes Simplex Virus Thymidine Kinase (HSV-TK),cytosine deaminase, Casp3, Casp8, Casp9, BAX, DFF40, cytosine deaminase,or other nucleic acid that encodes a protein that is capable of inducingapoptosis is a cell.

Apoptosis, or programmed cell death (PCD), is a common andevolutionarily conserved property of all metazoans. In many biologicalprocesses, apoptosis is required to eliminate supernumerary or dangerous(such as pre-cancerous) cells and to promote normal development.Dysregulation of apoptosis can, therefore, contribute to the developmentof many major diseases including cancer, autoimmunity andneurodegenerative disorders. In most cases, proteins of the caspasefamily execute the genetic programme that leads to cell death.

Apoptosis is triggered in a mammalian cell, in particular in a humancell, through the activation of caspase proteins, in particular thecaspase proteins CASP3, CASP8, and CASP9. See, for example, Xie et al.,Cancer Res 61(18):186-91 (2001); Carlotti et al., Cancer Gene Ther12(7):627-39 (2005); Lowe et al., Gene Ther 8(18):1363-71 (2001); andShariat et al., Cancer Res 61(6):2562-71 (2001).

DNA fragmentation factor (DFF) is a complex of the DNase DFF40 (CAD) andits chaperone/inhibitor DFF45 (ICAD-L). In its inactive form, DFF is aheterodimer composed of a 45 kDa chaperone inhibitor subunit (DFF45 orICAD), and a 40 kDa latent endonuclease subunit (DFF40 or CAD). Uponcaspase-3 cleavage of DFF45, DFF40 forms active endonucleasehomo-oligomers. It is activated during apoptosis to induce DNAfragmentation. DNA binding by DFF is mediated by the nuclease subunit,which can also form stable DNA complexes after release from DFF. Thenuclease subunit is inhibited in DNA cleavage but not in DNA binding.DFF45 can also be cleaved and inactivated by caspase-7 but not bycaspase-6 and caspase-8. The cleaved DFF45 fragments dissociate fromDFF40, allowing DFF40 to oligomerise, forming a large complex thatcleaves DNA by introducing double strand breaks. Histone H1 confers DNAbinding ability to DFF and stimulates the nuclease activity of DFF40.Activation of the apoptotic endonuclease DFF-40 is described in Liu etal., J Biol Chem 274(20):13836-40 (1999).

Thymidine kinase (TK) is an ATP-thymidine 5′-phosphotransferase that ispresent in all living cells as well as in certain viruses includingherpes simplex virus (HSV), varicella zoster virus (VZV), andEpstein-Barr virus (EBV). Thymidine kinase converts deoxythymidine intodeoxythymidine 5′-monophosphate (TMP), which is phosphorylated todeoxythymidine diphosphate and to deoxythymidine triphosphate bythymidylate kinase and nucleoside diphosphate kinase, respectively.Deoxythymidine triphosphase is incorporated into cellular DNA by DNApolymerases and viral reverse transcriptases.

When incorporated into DNA, certain dNTP analogs, such as syntheticanalogues of 2′-deoxy-guanosine (e.g., Ganciclovir), cause the prematuretermination of DNA synthesis, which triggers cellular apoptosis.

Within certain embodiments, the expression cassettes and systems of thepresent disclosure employ a nucleic acid that encodes HSV-TK. Followingthe administration to a human of a system employing a nucleic acidencoding HSV-TK, an analogue of a 2′-deoxy-nucleotide, such as2′-deoxy-guanosine, is administered to the human. The HSV-TK efficientlyconverts the 2′-deoxy-nucleotide analogue into a dNTP analogue, whichwhen incorporated into the DNA induces apoptosis in the target cell.

Cytosine deaminase (CD) catalyzes the hydrolytic conversion in DNA ofcytosine to uracil and ammonia. If a CD-modified site is recognized byan endonuclease, the phosphodiester bond is cleaved and, in a normalcell, is repaired by incorporating a new cytosine. In the presence of5-fluorocytosine (5-FC), cytosine deaminase converts 5-FC into5-fluorouracil (5-FU), which can inhibit target cell growth. Transgenicexpression of CD in a target cell, therefore, reduces the growth and/orsurvival of the target cell.

The present disclosure provides expression constructs and systems thatfurther comprise one or more safety features to ensure that theexpression of a nucleic acid encoding a therapeutic protein isupregulated in appropriate cells, over a desired time period, and/or toa specified level.

Within one such embodiments, expression constructs and systems of thepresent disclosure employ nucleic acids that encode inducible variantsof therapeutic proteins, including, for example, inducible variants ofCasp3, Casp8, Casp9, which require the further contacting of a cell withor administration to a human of a chemical or biological compound thatactivates the therapeutic protein.

Inducible suicide gene systems are well known and readily available inthe art and have been described, for example, in Miller et al., PCTPatent Publication No. WO 2008/154644 and Brenner, US Patent PublicationNo. 2011/0286980. In addition, Shah et al., Genesis 45(4):104-199 (2007)describe a double-inducible system for Caspase 3 and 9 that employsRU486 and chemical inducers of dimerization (CID). Straathof et al.,Blood 105(11):4247-4254 (2005) describe an inducible caspase 9 system inwhich caspase 9 is fused to a human FK506 binding protein (FKBP) toallow the conditional dimerization using the small molecule AP20187(ARIAD Pharmaceuticals, Cambridge, MA), which is a non-toxic syntheticanalog of FK506. Carlotti et al., Cancer Gene Ther 12(7):627-39 (2005)describe an inducible caspase 8 system by employing the ARIAD™homodimerization system (FKC8; ARIAD Pharmaceuticals).

Full-length inducible caspase 9 (F′F-C-Casp9.I.GFP) comprises afull-length caspase 9, including its caspase recruitment domain (CARD;GenBank NM001 229) linked to two 12 kDa human FK506 binding proteins(FKBP12; GenBank AH002 818) that contain an F36V mutation as describedin Clackson et al., Proc. Natl. Acad. Sci. U.S.A. 95:10437-10442 (1998)and are connected by a Ser-Gly-Gly-Gly-Ser linker that connects theFKBPs and caspase 9 to enhance flexibility.

In a further embodiment, the inducible suicide gene could be linked tothe nucleic acid sequence for a detectable biomarker such as luciferaseor green fluorescent protein to permit the detection of the targetedcells prior to administering a compound to activate an inducibletherapeutic protein.

Compositions and Formulations of Systems Comprising Vectors andExpression Cassettes

The present disclosure provides systems comprising a vector and anexpression cassette wherein the expression cassette comprises atranscriptional promoter that is responsive to one or more transcriptionfactors that are expressed in a target cell and a nucleic acid encodinga therapeutic protein. Systems can be administered to a human patient bythemselves or in pharmaceutical compositions where they are mixed withsuitable carriers or excipient(s) at doses to treat or ameliorate adisease or condition as described herein. Mixtures of these systems canalso be administered to the patient as a simple mixture or inpharmaceutical compositions.

Compositions within the scope of this disclosure include compositionswherein the therapeutic agent is a system comprising a vector and anexpression cassette in an amount effective to reduce or eliminate thegrowth and/or survival of a target cell such as a senescent cell, acancer cell, a cell infected with an infectious agent, a bacterial cell,or a cell that is associated with another disease or condition.Determination of optimal ranges of effective amounts of each componentis within the skill of the art. The effective dose is a function of anumber of factors, including the specific system, the presence of one ormore additional therapeutic agent within the composition or givenconcurrently with the system, the frequency of treatment, and thepatient's clinical status, age, health, and weight.

Compositions comprising a system may be administered parenterally. Asused herein, the term “parenteral administration” refers to modes ofadministration other than enteral and topical administration, usually byinjection, and include, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal, andintrasternal injection and infusion. Alternatively, or concurrently,administration may be orally.

Compositions comprising a system may, for example, be administeredintravenously via an intravenous push or bolus. Alternatively,compositions comprising a system may be administered via an intravenousinfusion.

Compositions include a therapeutically effective amount of a system, anda pharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skimmed milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents.

These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. Suchcompositions will contain a therapeutically effective amount of theinhibitor, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

Compositions can be formulated in accordance with routine procedures asa pharmaceutical composition adapted for intravenous administration to ahuman. Typically, compositions for intravenous administration aresolutions in sterile isotonic aqueous buffer. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lignocaine to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientsmay be mixed prior to administration.

The systems disclosed herein can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with anions suchas those derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, and the like, and those formed with cations such as those derivedfrom sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

Methods for Treatment of a Disease or Condition Associated with, and forReducing, Inhibiting, and/or Preventing the Growth and/or Survival of, aCell that is Associated with Aging, Cancer, Infectious Disease,Bacterial Infection, and/or Other Disease or Condition

The present disclosure provides methods for reducing, inhibiting, and/orpreventing the growth and or survival of a cell that is associated withaging, cancer, infectious disease, bacterial infection, and/or otherdisease or condition, which methods comprise contacting a target cell ora population of cell comprising a target cell with a system as describedherein, which system comprises a vector and an expression construct,which expression construct comprises a transcriptional promoter and anucleic acid.

The present disclosure also provides methods for the treatment of aging,cancer, infectious disease, bacterial infection, and/or other disease orcondition in a patient, which methods comprise the administration of asystem as described herein, which system comprises a vector and anexpression construct, which expression construct comprises atranscriptional promoter and a nucleic acid.

The present therapeutic methods involve contacting a target cell with,or administering to a human patient, a composition comprising one ormore system comprising a vector and an expression cassette to a humanpatient for reducing and/or eliminating the growth and/or survival of acell that is associated with senescence, cancer, an infectious disease,a bacterial infection or another disease or condition.

The amount of the system that will be effective in the treatment,inhibition, and/or prevention of aging, cancer, infectious disease,bacterial infection, or other disease or condition that is associatedwith the elevated expression of one or more transcription factors can bedetermined by standard clinical techniques. In vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

The systems or pharmaceutical compositions of the present disclosure canbe tested in vitro, and then in vivo for the desired therapeutic orprophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include the effect of a system ona cell line or a patient tissue sample. The effect of the system orpharmaceutical composition thereof on the cell line and/or tissue samplecan be determined utilizing techniques known to those of skill in theart including, but not limited to proliferation and apoptosis assays. Inaccordance with the present disclosure, in vitro assays that can be usedto determine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

The present disclosure provides methods for the treatment and growthand/or survival inhibition by administration to a subject of aneffective amount of a system or pharmaceutical composition thereof asdescribed herein. In one aspect, the system is substantially purifiedsuch that it is substantially free from substances that limit its effector produce undesired side-effects.

Methods of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The systems or compositions thereof may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the inhibitors or compositions into the centralnervous system by any suitable route, including intraventricular andintrathecal injection. Intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir. Pulmonary administration can also be employed,for example, by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

It may be desirable to administer the systems or compositions thereoflocally to the area in need of treatment; this may be achieved by, forexample, local infusion during surgery, topical application, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers.

The system can be delivered in a controlled release system placed inproximity of the therapeutic target, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release 2:115-138 (1984)).

Intravenous infusion of a compositions comprising a system may becontinuous for a duration of at least about one day, or at least aboutthree days, or at least about seven days, or at least about 14 days, orat least about 21 days, or at least about 28 days, or at least about 42days, or at least about 56 days, or at least about 84 days, or at leastabout 112 days.

Continuous intravenous infusion of a composition comprising a system maybe for a specified duration, followed by a rest period of anotherduration. For example, a continuous infusion duration may be from about1 day, to about 7 days, to about 14 days, to about 21 days, to about 28days, to about 42 days, to about 56 days, to about 84 days, or to about112 days. The continuous infusion may then be followed by a rest periodof from about 1 day, to about 2 days to about 3 days, to about 7 days,to about 14 days, or to about 28 days. Continuous infusion may then berepeated, as above, and followed by another rest period.

Regardless of the precise infusion protocol adopted, it will beunderstood that continuous infusion of a composition comprising a systemwill continue until either desired efficacy is achieved or anunacceptable level of toxicity becomes evident.

It will be understood that, unless indicated to the contrary, termsintended to be “open” (e.g., the term “including” should be interpretedas “including but not limited to,” the term “having” should beinterpreted as “having at least,” the term “includes” should beinterpreted as “includes but is not limited to,” etc.). Phrases such as“at least one,” and “one or more,” and terms such as “a” or “an” includeboth the singular and the plural.

It will be further understood that where features or aspects of thedisclosure are described in terms of Markush groups, the disclosure isalso intended to be described in terms of any individual member orsubgroup of members of the Markush group. Similarly, all rangesdisclosed herein also encompass all possible sub-ranges and combinationsof sub-ranges and that language such as “between,” “up to,” “at least,”“greater than,” “less than,” and the like include the number recited inthe range and includes each individual member.

All references cited herein, whether supra or infra, including, but notlimited to, patents, patent applications, and patent publications,whether U.S., PCT, or non-U.S. foreign, and all technical and/orscientific publications are hereby incorporated by reference in theirentirety.

EXAMPLES

While various embodiments have been disclosed herein, other embodimentswill be apparent to those skilled in the art. The various embodimentsdisclosed herein are for purposes of illustration and are not intendedto be limiting, with the true scope and spirit being indicated by theclaims. The present disclosure is further described with reference tothe following examples, which are provided to illustrate certainembodiments and are not intended to limit the scope of the presentdisclosure or the subject matter claimed.

Example 1 p14 FAST Fusogenic Lipid Nanoparticles (LNP) Enhance GeneDelivery to Tumors

This Example demonstrates that Fusogenix™ (Innovascreen, Halifax, NovaScotia, Canada) lipid nanoparticles utilizing a p14 FAST fusion fromreptilian reovirus are effective at delivering a plasmid DNA constructto a target tumor.

Fusogenix lipid nanoparticles labeled with ⁶⁴Cu (⁶⁴Cu NOTA-liposomes)either with or without a p14 FAST fusion protein (described in PCTPatent Publication Nos. WO2002044206A2 and WO2012040825A1) wereadministered intravenously to a M16 mouse model system for prostatecancer (PC3 cells). Seo, Bioconjug Chem 19(12):2577-2585 (2009) andReeves, Cancer Therapy 136(7):1731-1740 (2014). 24 hourspost-immunization, PC3 tumors were visualize using positron emissiontomography (PET). FIGS. 7A and 7B.

The data presented in FIG. 8 demonstrate a 50% increase in PC3 prostatetumor uptake of ⁶⁴Cu NOTA-liposomes with p14 FAST fusion protein ascompared to ⁶⁴Cu NOTA-liposomes without p14 FAST fusion protein. Thebiodistribution of labelled pegylated liposomes in nude mice expressedafter 24 hours is presented in FIG. 9 .

Example 2 In Vivo Administered p14 FAST Fusogenix Lipid Nanoparticlesare Non-Toxic and Well Tolerated

This Example demonstrates that Fusogenix™ (Innovascreen, Halifax, NovaScotia, Canada) lipid nanoparticles utilizing a p14 FAST fusion fromreptilian reovirus do not exhibit adverse side-effects in any of themajor mammalian organ systems examined when administered in vivo toSprague-Dawley rats. are effective at delivering a plasmid DNA constructto a target tumor.

Presented herein are comparative studies that were performed with N=20male rats treated with either (i) no LNPs (PBS), (ii) LNPs without p14,or (iii) p14 containing Fusogenix lipid nanoparticles (LNPs). Eachanimal received a total of three injections of 15 mg/kg in their tail,over a 4 day period. Treatment of the animals with p14 containing LNPsdid not result in any acute changes in animal behavior and animal growthwas not affected by treatment with p14 containing LNPs. Animals treatedwith p14 containing LNPs had similar organ weights as compared to allother animal groups studied.

Treatment with p14 containing LNPs did not affect the microscopicappearance of tissues from major organ systems. Tissues from the lungs,brain, heart, kidney, liver, reproductive organs, gut, endocrine system,lymph nodes, spleen, pancreas, bladder and tail were all independentlyexamined and p14 did not elicit any visible signs of toxicity.Importantly, the liver appeared to be unaffected by exposure to p14.Moreover, no differences were identified between the tissues of p14treated animals versus control groups.

A number of blood chemistry values were measured to determine the impactof p14 on physiological function and inflammation. Parameters such asALT and AST that denote acute liver function were all within normalranges. Fusogenix LNPs containing p14 do not show any adverseside-effects in any of the major mammalian organ systems examined.Histological appearance of tissues was also normal.

Mice were injected three (3) times at 10 day intervals with purified p14mixed with Freund's adjuvant. A first dose contained CFA (completeFreund's adjuvant) while second and third doses contained IFA(incomplete Freund's adjuvant). Each injection was with 50 μg of p14.Mice were sacrificed after 30 days and sera was analyzed for anti-p14antibodies. p14 lipid nanoparticles were also tested in two (2) mice viaintravenous injection of 400 μg of p53-iCasp9 Fusogenix lipidnanoparticles containing 240 μg of p14. Mice were sacrificed after 30days of injection and serum was analyzed for anti-p14 antibodies. Apositive control included purified antibodies spiked in serum at a highdose of 250 ng/ml and a low dose of 50 ng/ml. The data presented inFIGS. 10 and 11 demonstrate the safety and tolerability of Fusogenixlipid nanoparticles utilizing a reptilian reovirus p14 FAST fusionprotein. Anti-p14 and anti-LNP antibody assays demonstrated thatvirtually no antibody response was observed in immune competent mice(with and without adjuvant).

Ten (10) human serum samples were tested for Complementactivation-related psuedoallergy (CARPA) using C4d and iC3b complementELISA assays as described in Szebeni, Mol Immunol 61(2):163-73 (2014).The data presented in FIGS. 12 and 13 demonstrate that LNP formulationsaccording to the present disclosure were non-reactive with C4d (FIG. 12) and less reactive with iC3b (FIG. 13 ) as compared to Doxil in 8 outof 10 human samples (approximately 5-10% of humans exhibit a CARPAreaction to nanomedicines such as Doxil).

In vitro anti-p14 and anti-LNP antibody neutralization assays revealedthat vector neutralization required very high antibody concentrations.Moreover, vaccination or pretreatment with p14-LNPs did not result in adecrease in therapeutic efficacy and repeated in vivo dosing waseffective and well tolerated. CARPA assays with Fusogenix™ p14 FASTlipid nanoparticles elicit less complement activity as compared to acontrol pegylated liposomal doxorubicin (Doxil).

Example 3 In Vivo Suppression of p16-Positive Senescent Cell Burden inAged Mice

This Example demonstrates the target-cell specific suppression inp16-positive senescent cell burden following the in vivo administrationof an exemplary p16-targeting construct in an mouse model system foraging.

The aging mouse model exhibits a senescent cell burden (as defined bythe presence of p16⁺ cells) and secretion of factors associated with asenescence-associated secretory phenotype (SASP; van Deursen, Nature509(7501):439-446 (2014)).

A formulation comprising a vector and an expression construct, such as alipid nanoparticle (LNP) vector, e.g., a fusogenic LNP comprising afusogenic protein such as p14 FAST, encompassing a p16-iCasp9 expressionconstruct (pVAX1-16s-iCasp9; SEQ ID NO: 06; FIG. 16 ) which comprises anexemplary p16-targeting construct for the target cell-specificexpression of an inducible Caspase 9 (iCasp9) protein in target cellsexpressing p16, such as target cells that are associated with agingand/or senescence, which p16-targeting construct comprises a p16stranscriptional promoter in operable connection to iCasp9. or variantthereof expressing luciferase (for visualization), was administered invivo to an aged mouse via injection into a tail vein and theLNP+expression construct transfects target and non-target cells withoutspecificity. FIG. 19 . Upon subsequent in vivo administration of thechemical inducer of dimerization (CID), AP20187, p16+ target cells(e.g., senescent cells) underwent apoptosis, resulting in a reduction isSASP levels, while p16− cells remained viable.

Histological staining of senescent-associated β-gal in kidney cells froman in vivo aged mouse model either untreated (upper left panel) ortreated (upper right panel) following the in vivo administration (16animals at 80 weeks of age) of a formulation comprising a vector and anexpression construct, such as a lipid nanoparticle (LNP) vector, e.g., afusogenic LNP comprising a fusogenic protein such as p14 FAST,encompassing a p16-iCasp9 expression construct, e.g., pVAX1-16s-iCasp9or variant thereof, was administered in vivo to an aged mouse and kidneycells stained for β-gal. FIGS. 20A-D. The lower panel is aphotomicrograph of the histiological staining of senescent-associatedβ-gal in 4-month old kidney cells from a normal mouse. These datademonstrated a dose-dependent reduction of p16+ senescent kidney cells.

The dose-dependent targeting of p16+ kidney cells (FIG. 21 ), spleencells (FIG. 22 ), seminal vesicle cells (FIG. 23 ), inguinal fat cells(FIG. 24 ), and lung cells (FIG. 25 ) was demonstrated in naturally agedmice following the in vivo administration of a fusogenic lipidnanoparticle (LNP) formulation comprising a pVAX1-p16 expressionconstruct. Kidney cells were subjected to a qRT-PCR reaction to detectp16^(Ink4a) transcripts. Relative expression was calculated using 2ΔΔCt(Livak, Methods 25:402-408 (2001)).

Example 4 In Vivo Oncology Study with NSG Mice Implanted with a HumanProstate Tumor

This Example demonstrates the target-cell specific suppression ofp53-expressing prostate cancer cells in NSG mice implanted with a humanprostate tumor (i.e., a PC-3 xenograft).

Human prostate cancer PC-3 cells were treated with Fusogenix lipidnanoparticles carrying the pVax-p53-iCasp9-luc (luciferin) plasmid (inthe presence and absence of the homodimerizer AP201870) and assessed foriCasp9 expression and subjected to Western blot analysis of iCasp 9 andCasp 9 protein levels obtained with p53-expressing cells (pVax-p53) andcontrol cells (pcDNA3-GFP). FIG. 36 . These data demonstrated that theaddition of the chemical inducer of dimerization (CID; e.g., AP20187 andAP1903) abolishes the expression of iCasp9 and luciferase inp53-expressing cells engineered to express iCasp9 or luciferase.

Human prostate cancer cells (LNCaP (FIG. 38 ), DU145 (FIG. 39 ), andPC-3 (FIG. 40 )) and normal epithelial cells (RWPE (FIG. 41 )) weretreated with Fusogenix lipid nanoparticles carrying thepVax-p53-iCasp9-luc plasmid and assessed for iCasp9 expression byWestern blot and luminescence assays. These data demonstrated thataddition of the chemical inducer of dimerization (CID; e.g., AP20187 andAP1903) abolished the expression of iCasp9 and luciferase inp53-expressing cells engineered to express iCasp9 or luciferase.

Human prostate cancer PC-3 cells were treated with Fusogenix lipidnanoparticles carrying the pVax-p53-iCasp9-luc (luciferin) plasmid (inthe presence and absence of the homodimerizer AP20187) and assessed foriCasp9 expression. The data presented in FIG. 42 demonstrated that theaddition of the chemical inducer of dimerization (CID; e.g., AP20187 andAP1903) abolished the expression of iCasp9 and luciferase inp53-expressing cells engineered to express iCasp9 or luciferase.

Flow cytometry apoptosis data (Annexin V) from human prostate cancerPC-3 cells treated with pVax-p53 Fusogenix lipid nanoparticles (in theabsence and presence of AP20187, FIGS. 43A and 44A and 43B and 44B,respectively) demonstrated that suicide gene therapy selectively killedp53-expressing human prostate cancer cells in culture by inducingapoptosis (Luciferase-Annexin V flow cytometry).

A pre-clinical oncology study according to the present disclosure wasconducted with 30×NSG mice implanted with human prostate tumor cells.FIG. 45 . NSG mice bearing a subcutaneous human prostate PC-3 tumor wereinjected intratumorally (IT) with 100 μg Fusogenix pVax-p53 formulation,followed 96 hours later by intravenous (IV) administration of 2 mg/kg ofthe homodimerizer AP20187. FIG. 46 . Tumors from the NSG mice bearingsubcutaneous human prostate PC-3 tumors injected intratumorally with 100μg Fusogenix pVax-p53 formulation, followed 96 hours by 2 mg/kg AP20187IV, were photographed (FIGS. 47A-47C).

Four NSG mice bearing subcutaneous human prostate cancer PC-3 tumorsthat were injected intravenously (IV) with 4×100 μg doses of FusogenixpVax-p53 formulation, followed 24 hours later by 2 mg/kg AP20187 IV.Tumor volume was measured and plotted as a function of time following IVinjection. FIGS. 48-51 .

The percentage change in tumor volume was determined and plotted as afunction of time after in vivo administration of a chemical inducer ofdimerization (CID) in NSG mice (N=6 for all groups) bearing a prostatetumor that were treated with intravenous p14 LNP pVAX. FIG. 52 . Thepercent survival was determined and plotted as a function of time afterin vivo administration of a chemical inducer of dimerization (CID) inNSG mice (N=6 for all groups) bearing a prostate tumor that were treatedwith intravenous p14 LNP pVAX. FIG. 53 .

A dose escalation study was carried out in which the percentage changein tumor volume as a function of time after in vivo administration of achemical inducer of dimerization (CID) in NOD-SCID mice (N=6 for allgroups) bearing a prostate tumor that were treated with 100 μg, 400 μg,and 1000 μg of intravenous p14 LNP pVAX. NOD-SCID mice were implantedsubcutaneously with 500,000 PC-3 cells and randomized into treatmentgroups when their tumors reached 200 mm³, (N=2 for all groups). Animalswere injected with their assigned dose of p53-iCasp9 LNP IV twicefollowed by 2 mg/kg dimerizer. Tumors were measured directly every 24hours. FIG. 54 .

In total, the data presented herein demonstrate that apoptosis can bereliably induced in a p53+ prostate cancer cell-specific manner by theintravenous administration of fusogenic lipid nanoparticle formulationscomprising a p53-iCasp9 expression construct.

Example 5 In Vivo Suppression of Metastases in NOD-SCID Mice Implantedwith a Metastatic Tumor

The suppression of metastatic tumor growth with repeat treatment of ap53-iCasp9 LNP with or without a chemical inducer of dimerization (CID)was demonstrated in a NOD-SCID mouse model system.

NOD-SCID mice were injected with 500,000 PC-3M-luciferase cells on Day0, LNP dosing was started on Day 22 with 150 μg p53-iCasp9 LNP.Dimerizer doses started Day 24 at 2 mg/kg. Mice were imaged every 24-48hours to detect whole animal luminescence. FIG. 55 .

Example 6 In Vivo Suppression of Melanoma in Isogenic C57B6 MiceImplanted with B16 Murine Melanoma Cells

Isogenic C57B6 mice implanted with B16 murine melanoma cells weretreated with LNPs containing a construct encoding iCasp9 and murineCD40L under control of the murine p53 promoter followed by the AP20187dimerizer.

The percentage change in tumor volume (FIG. 56 ) and percent survival(FIG. 57 ) mas measured as a function of time after in vivoadministration of a chemical inducer of dimerization (CID) in isogenicC57B6 mice implanted by subcutaneous injection with 250,000 B16 murinemelanoma cells treated (grown to 400 mm³) with LNPs containing aconstruct encoding iCasp9 and murine CD40L under control of the murinep53 promoter.

These data demonstrated that, even though the rapid (10 hour) doublingtime of the B16 cells made them largely refractory to the iCasp9-inducedapoptosis, they still secreted enough CD40L to effectively halt thetumor's growth. A construct encoding GMCSF+OVA antigen was also testedand determined to be more effective than iCasp9 alone, but lesseffective than the CD40L version. N=3 for both groups.

Example 7 In Vivo Suppression of Lung Cancer Metastasis in MiceImplanted with B16F10 Murine Melanoma Cells

This Example demonstrates the in vivo p53+ target cell suppressionmurine p53+B16F10 melanoma target cells implanted in a lung metastasismouse model system.

A B16F10 lung metastasis model system was employed in which 100 μg of acontrol LNP or a p53-iCasp9 LNP was administered intravenously at days3, 6, 9, and 12 following the intravenous injection of 75,000 B16F10cells. At days 5, 8, 11, and 13, a chemical inducer of dimerization(CID) was administered intraperitoneally. Animals were sacrificed at day14 and lung metastases were quantified. FIGS. 58A-58D and 59 .

1-35. (canceled)
 36. A lipid-based nanoparticle (LNP) formulation fortargeted production of a therapeutic protein within target cells, theLNP formulation comprises: a. a lipid nanoparticle vector comprising1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at a molar ratio of22.5-37.5 mole %; and b. an expression construct, wherein the expressionconstruct is configured for preferential production of the therapeuticprotein within the target cells, wherein the expression constructcomprises: i. a transcriptional promoter that is activated in responseto one or more factors that are preferentially produced within thetarget cells as compared to non-target cells; and ii. a nucleic acidthat is operably linked to and under regulatory control of thetranscriptional promoter, wherein the nucleic acid encodes thetherapeutic protein, wherein the therapeutic protein is capable ofreducing growth or survival of the target cells, wherein the therapeuticprotein is produced within the target cells but is substantially notproduced in the non-target cells.
 37. The LNP formulation of claim 36,wherein the lipid nanoparticle vector further comprises1,2-dioleoyl-3-dimethylammonium-propane (DODAP).
 38. The LNP formulationof claim 37, wherein the lipid nanoparticle vector comprises the DODAPat a molar ratio of at least 35 mole %.
 39. The LNP formulation of claim36, wherein the lipid nanoparticle vector comprises the DOPE at a molarratio of about 30 mole %.
 40. The LNP formulation of claim 36, whereinthe expression construct is present in the LNP formulation at aconcentration ranging from 20 μg/mL to 1.5 mg/mL.
 41. The LNPformulation of claim 36, wherein the transcriptional promoter is a p16transcriptional promoter or a p53 transcriptional promoter.
 42. The LNPformulation of claim 36, wherein the transcriptional promoter is a p16transcriptional promoter.
 43. The LNP formulation of claim 36, whereinthe therapeutic protein is selected from the group consisting of acaspase (Casp), an inducible caspase (iCasp), a self-activating caspase(saCasp), BAX, DFF40, HSV-TK, and cytosine deaminase.
 44. The LNPformulation of claim 36, wherein the therapeutic protein is a caspase.45. The LNP formulation of claim 36, wherein the therapeutic protein isa Casp3, a Casp8, or a Casp9.
 46. The LNP formulation of claim 36,wherein the therapeutic protein is Casp9.
 47. The LNP formulation ofclaim 36, wherein the therapeutic protein is an inducible Casp9(iCasp9).
 48. The LNP formulation of claim 36, wherein the therapeuticprotein is a self-activating Casp9 (saCasp9).
 49. The LNP formulation ofclaim 36, wherein the lipid nanoparticle vector further comprises afusogenic protein.
 50. The LNP formulation of claim 49, wherein thefusogenic protein comprises a fusion-associated small transmembrane(FAST) protein.
 51. The LNP formulation of claim 49, wherein thefusogenic protein comprises an ectodomain amino acid sequence from afirst reovirus FAST protein and an endodomain amino acid sequence from asecond reovirus FAST protein.
 52. The LNP formulation of claim 49,wherein the fusogenic protein comprises an ectodomain amino acidsequence with at least 80% sequence identity to an endodomain of a p14FAST protein and an endodomain amino acid sequence with at least 80%sequence identity to a p15 FAST protein.
 53. The LNP formulation ofclaim 49, wherein the fusogenic protein comprises the amino acidsequence of SEQ ID NO:
 17. 54. A lipid-based nanoparticle (LNP)formulation for targeted production of a therapeutic protein withintarget cells, the LNP formulation comprising: a. a lipid nanoparticlevector comprising 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol (DMG-PEG) at a molar ratio of 3-5 mole %; and b. an expressionconstruct, wherein the expression construct is configured forpreferential production of the therapeutic protein within the targetcells, wherein the expression construct comprises: i. a transcriptionalpromoter that is activated in response to one or more factors that arepreferentially produced within the target cells as compared tonon-target cells; and ii. a nucleic acid that is operably linked to andunder regulatory control of the transcriptional promoter, wherein thenucleic acid encodes the therapeutic protein, wherein the therapeuticprotein is capable of reducing growth or survival of the target cells,wherein the therapeutic protein is produced within the target cells butis substantially not produced in the non-target cells.
 55. The LNPformulation of claim 54, wherein the lipid nanoparticle vector furthercomprises 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).
 56. The LNPformulation of claim 55, wherein the lipid nanoparticle vector comprisesthe DODAP at a molar ratio of at least 35 mole %.
 57. The LNPformulation of claim 54, wherein the lipid nanoparticle vector comprisesthe DMG-PEG at a molar concentration of about 4 mole %.
 58. The LNPformulation of claim 54, wherein the expression construct is present inthe LNP formulation at a concentration ranging from 20 μg/mL to 1.5mg/mL.
 59. The LNP formulation of claim 54, wherein the transcriptionalpromoter is a p16 transcriptional promoter or a p53 transcriptionalpromoter.
 60. The LNP formulation of claim 54, wherein thetranscriptional promoter is a p16 transcriptional promoter.
 61. The LNPformulation of claim 54, wherein the therapeutic protein is selectedfrom the group consisting of a caspase (Casp), an inducible caspase(iCasp), a self-activating caspase (saCasp), BAX, DFF40, HSV-TK, andcytosine deaminase.
 62. The LNP formulation of claim 54, wherein thetherapeutic protein is a caspase.
 63. The LNP formulation of claim 54,wherein the therapeutic protein is a Casp3, a Casp8, or a Casp9.
 64. TheLNP formulation of claim 54, wherein the therapeutic protein is Casp9.65. The LNP formulation of claim 54, wherein the therapeutic protein isan inducible Casp9 (iCasp9).
 66. The LNP formulation of claim 54,wherein the therapeutic protein is a self-activating Casp9 (saCasp9).67. The LNP formulation of claim 54, wherein the lipid nanoparticlevector further comprises a fusogenic protein.
 68. The LNP formulation ofclaim 67, wherein the fusogenic protein comprises a fusion-associatedsmall transmembrane (FAST) protein.
 69. The LNP formulation of claim 67,wherein the fusogenic protein comprises an ectodomain amino acidsequence from a first reovirus FAST protein and an endodomain amino acidsequence from a second reovirus FAST protein.
 70. The LNP formulation ofclaim 67, wherein the fusogenic protein comprises an ectodomain aminoacid sequence with at least 80% sequence identity to an endodomain of ap14 FAST protein and an endodomain amino acid sequence with at least 80%sequence identity to a p15 FAST protein.